DOC-蛋白质化学-研究生课程(英文版)(doc114)-石油化工

DOC-蛋白质化学-研究生课程(英文版)(doc114)-石油化工 Protein Chemistry Postgraduate students’ course CONTENTS Chapter 1 Introduction to Protein Chemistry 1.1 Overview—Advances in Protein Chemistry 1.2 General Concept of Protein 1.3 Protein Functions 1.4 Protein Structures 1.4.1 The Building Blocks of Proteins 1.4.2 Polypeptide Chain 1.4.3 Conformation of Polypeptide Chain 1.4.4 Protein Structure Biology 1.5 Interactions of Protein with Other Molecules 1.6 Protein Engineering 1.7 Proteome and Proteomics 1.8 International Journals and Websites Related to Protein Chemistry Chapter 2 Physic-chemical Properties of Proteins 2.1 Size and Shape of Protein Molecules 2.2 The Charge Properties of Protein Molecules 2.2.1 The Charge Properties of Amino Acids 2.2.2 The Charge Properties of Protein Molecules 2.2.3 Isoelectric Point of Proteins 2.2.4 Electrophoresis 2.2.5 Ion Exchange 2.3 Colloid Property of Proteins 2.3.1 Diffusion 2.3.2 Viscosity 2.3.3 Impermeability 2.4 Precipitation 2.4.1 Salting out and Salting in 2.4.2 Basic and Acidic precipitation 2.4.3 Organic Solvent-caused precipitation 2.4.4 Heavy metal-caused precipitation 2.4.5 Interaction of Antibody and Antigen 2.4.6 Others 2.5 Color-formation of Proteins 2.6 Spectro-properties of Proteins 2.6.1 Ultraviolet absorption 2.6.2 Fluorescence spectrum 2.6.3 Circular Dichroism Spectrum Chapter 3 Methods for Characterization and Purification of Proteins 3.1 Methods of Protein Characterization 3.1.1 Solubility Reflects a Balance of Protein-Solvent Interaction 3.1.2 Several Methods are Available for Determina- tion of Gross Size and Shape 3.1.3 Electrophoretic Methods are the Best Way to Analyze Mixtures 3.2 Methods for Protein Purification 3.2.1 Differential Centrifugation Subdivides Crude Extracts into Two or More Fractions 3.2.2 Differential Precipitation is Based on Solubility Differences 3.2.3 Column Procedures Are the Most Versatile Purification Methods 3.2.4 Electrophoretic Methods are Used for Preparation and Analysis 3.2.5 Purification of Specific Proteins Involves Combination of Different Procedures Chapter 4 The Building Blocks of Proteins---- Amino Acids, Peptides, and Polypeptides 4.1 Aminao acids 4.1.1 Amino acids have both acid and base properties 4.1.2 Aromatic amino acid absorb light in the near- ultraviolet 4.1.3 All amino acids except glycine show asymetry 4.2 Peptides and polypeptides 4.3 Determination of amino acid composition of proteins 4.4 Determination of amino acid sequence of proteins 4.5 Chemical synthesis of peptides and polypeptides Chapter 5 The Three-Dimensional Structures of Proteins 5.1 The information for folding is contained in the primary structure 5.2 The Ramachadran Plot predicts sterically permissible strucutre 5.3 Protein folding reveals a hierarchy of structural organization 5.4 Two secondary structure are found in most proteins 5.4.1 The , helix 5.4.2 The , Sheet 5.5 Pauling and Corey provided the foundation for understanding of fibrous protein structure 5.6 Collagan forms a unique triple-stranded structure 5.7 In globular proteins, secondary structure element are connected in simple motifs 5.8 The domain is the basic unit of tertiary structure 5.8.1 The helix-loop-helix motif is the basic component found in , domain structures 5.8.2 ,/, domains exploit the ,-,-, motif 5.8.3 Antiparalle , domains show a great variety of topologies 5.8.4 Some proteins or domains require additional features to account for their stability 5.8.5 Many proteins contain more than one domain 5.9 Quaternary structure depends on the interaction of two or more proteins or protein subunits 5.10 Predicting protein structure from protein prmary structure 5.11 Methods for determing protein conformation 5.11.1 X-ray diffraction analysis of fibrous proteins 5.11.2 X-ray diffraction analysis of proteins crystals 5.11.3 Nuclear magnetic resonance (NMR) complements X-ray crystallography 5.11.4 Optical Rotatory Dispersion (ORD) and Circular Dichroism (CD) Chapter 6 Protein Structure Prediction Reference: ―Protein Structure Prediction A Practical Approach‖ edited by M J E Sternberg IRL Press Chapter 7 Protein Folding and Unfolding 7.1 Kinetic Analysis of Complex Reactions 7.2 Kinetics of Unfolding 7.3 Kinetics of Refolding 7.3.1 Peptide bond isomerization 7.3.2 Refolding in the absence of slow peptide bond isomerization 7.3.3 The prefolded state 7.3.4 The transition state for folding 7.4 Folding Pathways 7.4.1 Trapping intermediates with disulfides 7.4.2 Disulfide folding pathway of BPTI 7.5 Folding of large proteins 7.6 Biosynthetic folding 7.6.1 Basic methods 7.6.2 Multiple phases and cis-peptidyl-prolyl bonds Chapter 8 Stabilizing Protein Function 8.1 Understanding Protein Stability 8.1.1 Protein Stability and Its Measurement Introduction Protein Structure Definition and measurement of protein stability Folding stability Kinetic stability 8.1.2 Studies on Denaturation, Inactivation and Stabilizing Interactions Introduction Denaturation studies Detelerious chemical reactions in proteins Deamidation of asparagine residues Isomerization of prolines Destructive oxidation events Proteolytic processes Probing the stabilizing interactions in proteins Replacement of conserved residues: E. coli Thioredoxin Carbohydrate side chains and protein stability Is there a trade-off between stability and Activity ? 8.1.3 Enzymes in Organic Media Introduction Enzyme behavior in anhydrous organic solvents Some case studies Water- Chapter 9 Functional Diversity of Proteins 9.1 Targeting and functional diversity 9.1.1 Proteins are directed to the regions where they are utilized 9.1.2 Classification of proteins according to location emphasizes functionality 9.1.3 Protein structure is suited to protein function 9.2 Hemoglobin—an allosteric oxygen-binding protein 9.3 Muscle---an aggregate of proteins involved in contraction 9.4 Protein diversification as a result of evolutionary pressures Chapter 10 Proteins in Solution and in Membranes 10.1 Introduction 10.2 Physical and chemical properties of soluble proteins 10.2.1 Aqueous solubility 10.2.2 Hydrodynamic properties in aqueous solution 10.2.3 Spectral properties 10.2.4 Ionization 10.2.5 Chemical properties 10.3 Proteins in membranes 10.3.1 Association with membrane 10.3.2 Structures of integral membrane proteins 10.3.3 Identifying amino acid sequences likely to transverse membranes 10.3.4 Dynamic behavior in membranes 10.4 Flexibility of protein structure Chapter 11 Protein Engineering 11.1 Introduction to Protein Engineering 11.2 Production and Analytical Characterization of Proteins 11.2.1 DNA level processes Finding the protein of interest Developing recombinant DNA libaries Mutagenesis principle 11.2.2 Protein Characterization Methods to determine and assess the protein strucutre and composition Assessment of mutant proteins 11.2.3 Summary of Issues to Consider before Engineering a Protein What is needed? Industrial issues 11.3 Protein Engineering for Stability 11.4 Engineering therapeutic antibody 11.5 Site-directed drug design Chapter 12 Proteome and Proteomics 12.1 Introduction to the Proteomics 12.1.1 Proteome: a new wod, a new field biology 12.1.2 The Proteome and Technology Thinking in two dimenesions Further dimensions in protein analysis Information and the proteome 12.2 Two-Dimensional Electrophoresis: The State of the Art and Future Direction 12.3 Protein Identification in Proteome Projects 12.4 The Importance of Protein Co- and Post- Translational Modifications in Proteome Projects 12.5 Proteome Databases 12.6 Interfacing and Integrating Databases 12.7 Large-scale Comparative Protein Modelling 12.8 Applications of Proteomics Chapte 13 Protein Synthesis, Targeting, and Turnover 13.1 The cellular machinery of protein synthesis 13.1.1 Messenger RNA is the template for protein synthesis 13.1.2 Transfer RNAs order activated amino acids on the mRNA template 13.1.3 Ribosomes are the site of protein synthesis 13.2 The Genetic code 13.2.1 The code was deciphered with the help of synthetic messengers 13.2.2 The code is highly degenerate 13.2.3 Wobble introduces ambiguity into codon-anticodon interactions 13.2.4 The code is not universal 13.2.5 The rules regarding codon-anticodon pairing are species-specific 13.3 The Steps in translation 13.3.1 Chapter 1 Introduction to Protein Chemistry 1.1 Overview—Advances in Protein Chemistry 1. Advances in technological and experimental approaches. Experimentalists are able to alter the activity and stability of proteins by protein engineering, and the first tentative steps in protein design are under way. 2. Theoreticians are able to simulate many aspects of folding and catalysis with increasing detail and reliability. 3. The ultimate goal of protein science is to be able to predict the structure and active of a protein de novo and how it will bind ligands 4. Proteome and proteomics Protein biology is the study of protein structure revealing details of the function and life cycle of individual polypeptides. Completion of the sequences of animal and plant genomes will not be the consummation of modern biology, but a new beginning. The experimental and philosophical challenge is not just to identify polypeptide gene products for all coding genes, but to provide a rationale for how they work. As part of a cellular response to external stimuli, proteins may be altered by chemical modification or change of subcellular localization, processing, degradation, or concentration. How do proteins work? How they work together? How do they work over time and space? The development of technologies and experimental approaches that were required to answer the questions of protein biology accelerated during the early period of genomic analysis. These technologies are now essential tools of experimental biology. All of the strategies aim toward the highest sensitivity analysis possible. Chromatographic and electrophoretic separation and determination, Edman sequence analysis, amino acid analysis, and mass spectrometry are now all performed routinely with a few picomoles of protein or peptide. Subpicomoles and low femtomole levels of analysis are reported in the literature more frequently, and experiments detecting attomoles of protein or peptide have been described. Covalent modifications of proteins are an essential part of the language of intracellular and intercellular communication. They may be reversible or irreversible. They may be required for biological activity, or simply modulate it, functioning as ―molecular switches‖. They are important for signalling and molecular and cellular recognition. These modifications may impart structural stability or unique structural features, facilitate protein folding, or promote intermolecular interactions. They may anchor a protein in a membrane or determine intracellular or extracellular position. Modification can alter the biological lifetime of a protein, and define the process by which it is degraded. Whereas some modifications are present on an entire population of a polypeptide, others are present on only a small subset of the protein at any one moment. Quantification of the extent and range of modification present in this population is not routinely analyzed, but could have important biological implications. Thus, knowledge of the possible modifications of a particular protein, as well as modifications induced by external stimuli, are of fundamental importance to research in structural biology and cell biology alike. Protein biology also includes the design of new proteins to test biological and physiological hypothesis. One can now design and prepare novel protein with new functions that cannot be isolated from nature. How can one manipulate proteins to understand their original function or to make them perform new ones? On what principles can these designs be based? Both molecular biological and chemical synthetic procedures have made it possible to alter polypeptides structure to probe details of function. Strategies can be developed to alter a single amino acid, to substitute a non-natural residue, or to mix and match functional units to create new biological entities. Last, but not the least, the bio-informatics has accelerated the development of protein study. 1.2 General Concept of Protein (see also page 1 in Text) 1.3 Protein Functions 1.4 Protein Structures 1.4.1 The Building Blocks of Proteins 1.4.2 Polypeptide Chain 1.4.3 Conformation of Polypeptide Chain 1.4.4 Protein Structure Biology 1.5 Interactions of Protein with Other Molecules 1.6 Protein Engineering 1.7 Proteome and Proteomics Proteome indicates the PROTEins expressed by a genOME or tissue. Despite being first used in late 1994 at the Siena 2-D electrophoresis meeting, the term proteome is already widely accepted. Zit is mentioned in more than 30 papers (till the end of 1997), including those from Science and Nature, and ―Proteomics: has been the subject of a rash of conference in 1997. The antecedent for proteome is genome, itself a relative new word. Recently, genome has become a generic term for ―big science‖ molecular biology. People think of grand dreams like the human genome project in the context of genome. And the genome projects have captured the imagination of scientific funding bodies and the biotechnology industry. By sequencing the entire genome of an organism, here for the first time in biology is the complexity of an organism understood at the level of information content. The proteome, unlike the genome, is not a fixed feature of an organism. Instead, it changes with the state of development, the tissue or even the environmental conditions under which an organism finds itself. There are therefore many more proteins in a proteome than gens in a genome, especially for eukaryotes. This is because there can be various ways a gene is spliced in constructing mRNA, and there are many ways that the same protein can be post-translationally altered. So one of the famous dogmas of biology, the one-gene-one-enzyme hypothesis of Beadle and Tatum, is no longer tenable. 1.8 International Journals and Websites Related to Protein Chemistry Table 1.1 International Journals Related to Protein Chemistry (295 Journals in Bichemistry and Molecular Biology in 1998) Rank Journal Abbreviation Impact 1998 articles Factor 1 Annu Rev Biochem 39.000 26 2 Cell 38.686 407 8 Nat Struc Biol 13.563 158 15 Curr Opin Struc Biol 8.690 93 23 J Biol Chem 7.199 4879 31 Adv Protein Chem 5.870 7 45 Biochemistry 4.628 2014 46 Protein Sci 4.440 277 52 Enzyme Protein 4.080 66 Proteins 3.346 177 73 Protein Eng 2.947 160 77 Method Enzymol 2.823 218 95 Arch Biochem Biophys 2.497 481 97 Biochim Biophys Acta 2.478 1813 123 Anal Biochem 1.991 460 132 Adv Enzyme Regul 1.884 22 167 Protein Expres Purif 1.382 166 175 J Protein Chem 1.278 140 Table 1.2 Websites Related to Protein Chemistry GenBank (美国基因、蛋白数据库) GeneCard() EMBL (欧洲分子生物学实验室数据库) HUGD(人类基因突变数据库) DDBJ(日本国家遗传研究所基因数据库) Swiss-Port(瑞士蛋白数据库) 蛋白三维结构数据库(美国Brookheaven实验室) PIR(美国国家生物医学技术研究基金会蛋白数据库) Human SNP Database(Whitehead institute/MIT center for genome research) OPD (Oligonucleotide probe database) PROSITE (EMBL，蛋白序列中的特征序列及位点) HSSP(EMBL, 三维结构已知的蛋白的同源蛋白) DSSP(EMBL,蛋白二级结构及溶剂信息) FSSP(EMBL，蛋白折叠方式相似性的结构家族) SBASE(ABC,ICGEB，蛋白结构域、功能域资料) TFD(NCBI，各种转录因子及其特性) TRANSTERM(新西兰Otago大学, 翻译终止信号数据库) Rebase(New england biolabs公司，限制酶及甲基化酶数据库) Genome Data Base (GDB) The PredictProtein server(EMBL in Heidelberg) BLAST () dbEST() Entrez () MMBD() 基因芯片数据库---? MPDB(合成寡核苷酸探针数据库)---? Chapter 2 Physic-chemical Properties of Proteins Chapter 3 Methods for Characterization and Purification of Proteins To analyze the structure of a protein, we must isolate it from the complex mixture of substances in which it exists in whole cell. The primary object of this chapter is to describe techniques and strategies used for protein purification. Because these procedures are often used for protein characterization as well, they will add to the methods already discussed for protein characterization. 3.1 Methods of Protein Characterization First we will discuss methods used for protein characterization. Some of them will be discussed in later related chapters. 3.1.1 Solubility Reflects a Balance of Protein-Solvent Interaction The solubility of a protein reflects a delicate balance between different energetic interactions, both internally within the protein and between the protein and the surrounding solvent. Consequently, the protein‘s solvent or thermal environment could affect both its solubility and structure. As we have seen, extreme changes can lead to denaturation. In this part, we will, for the most part, be concerned with conditions under which the native structure is maintained. Changes in protein solubility that do not destroy the molecule‘s strucutral integrity can occur in several ways. A. Minimum in solubility occurs at the isoelectric point Proteins typically have on their surfaces charged amino acid chains that undergo energetically favorable polar interactions with the surrounding water. The total charge on the protein is the sum of the side-chain charges. However, the actual charge on the weakly acidic and basic side-chain groups also depends on the solution pH. The decrease in solubility at the isoelectric pH reflects the fact that the individual protein molecules, which would all have a similar charge at pH values away from their isoelectric points, cease to repel each other. B. Salting in and salting out Proteins also show a variation in solubility that depends on the concentration of salts in the solution. These frequently complex effects may involve specific interactions between charged side chains and solution ion, or, particularly at high salt concentrations, may reflect more comprehensive changes in the solvent properties. Figure 7.1 and 7.2 Salting-in effect: The effect of salts such as sodium chloride on increasing the solubility of globulins is often referred to as salting in. The salting in effect is related to the nonspecific effect the salt has on increasing the ionic strength of the solution. The higher the ionic strength, the smaller are the interactions between charged groups on the same or different proteins. Salting-out effect: The effect of salt such as ammonium sulfate on decreasing the solubility of proteins is refered to as salting out, which occurs with salts that effectively compete with the protein for available water molecules. In this case the protein molecules tend to associate with each other because at high salt concentrations, protein- protein interactions become energetically more favorable than protein-solvent interactions. Each protein has a characteristic salting-out point, and we can exploit this fact to make protein separations in crude extracts. 3.1.2 Several Methods are Available for Determina- tion of Gross Size and Shape Several methods are available for determining the size and shape of protein molecules in solution. A. Sedimentation rates is a function of size and shape Information concerning the molecular weight of a protein can be obtained by observing its behavior in an intense centrifugal field. To get a qualitative understanding of how this method works, we must first recognize that protein molecules are generally slightly denser than water. However, the molecules in a protein solution seldom settle out in the earth‘s gravitational field(1，g) because they are constantly being stirred up by collisions with surrounding solvent molecules. Nevertheless, protein molecules in solution can be made to settle if they are subjected to very high centrifugal force field(~100000，g), such as can be attained in an ultracentrifuge. The protein molecules slowly migrate toward the bottom of the centrifuge tube at a rate that proportional to their molecular weight. B. Gel-exclusive chromatography gives a measure of size Gel-exclusive chromatography is used for size estimation as well as protein purification. This popular technique exploits the availability of both natural polysaccharide and synthetic polymers that can be formed into beads with varying pore sizes, depending on the extent of cross-linking between polymer chains. Figure 7.5 and 7.6 3.1.3 Electrophoretic Methods are the Best Way to Analyze Mixtures Electrophoresis is one of the most commonly used techniques in biochemistry. Electrophoresis is very much like sedimentation, since in both cases a force gradient leads to protein transport in the direction of the force. In the case of sedimentation the force is gravity, so the rate of migration depends on the effective mass of the particle. In electrophoresis the force is the electrical potential, E, so the rate of migration depends on the net charge on the molecule, rather than its mass. (Figure 7.7 and 7.8). (1) SDS-PAGE (2) IEF-SDS-PAGE 3.2 Methods for Protein Purification Before we can fully characterize a protein, we must purify it from a natural source. Once the decision has been made to purify a particular protein, several factors must be weighed. e.g. how much material is needed? What level of purity is required? The starting material should be readily available and should contain the desired protein in relative abundance. If the protein is part of a large structure, such as nucleus, the mitochondria, or the ribosome, then it is advisable to isolate the large structure first from a crude cell extract. Purification must usually be performed in a series of steps, using different techniques at each step. Some purification techniques are more useful when handling large amounts of material, whereas others work best on small amounts. A purification procedure is arranged so that the techniques that are best for work with large amounts are during early steps in the overall purification. The suitability of each purification step is evaluated in terms of the amount of purification achieved by that step and the percent recovery of the desired protein. Combining techniques introduces new considerations and new problems. If each of two purification techniques gives a ten-fold enrichment for the desired protein when executed independently on a crude extract, this does not mean that they will give 100-fold enrichment when combined. In general, they will give somewhat less. As a rule, purification techniques that combine most effectively usually are based on different properties of the protein. Throughout the purification we must have a convenient means of assaying for the desired proteins so that we can know the extent to which it is being enriched relative to the other proteins in the starting material. In addition, a major concern in protein purification is stability. Once the protein is removed from its normal habitat, it becomes susceptible to a variety of denaturation and degradation reactions. Specific inhibitors are sometimes added to minimize attack by proteases on the desired protein. During purification it is usual to carry out all operations at 5C or below. In their natural habitat, proteins are usually surrounded by other protein and organic factors. When these are removed or diluted, as during purification, the protein becomes surrounded by water on all sides. Proteins react differently to a pure aqueous environment; many are destabilized and rapidly denatured. A common remedial measure is to add 5% to 20% glycerol to the purification buffer. The organic surface of the glycerol is believed to simulate the environment of the protein in the intact cell. Two other ingredients that are most frequently added to purification buffers are mercaptoethanol and EDTA. 3.2.1 Differential Centrifugation Subdivides Crude Extracts into Two or More Fractions 3.2.2 Differential Precipitation is Based on Solubility Differences 3.2.3 Column Procedures Are the Most Versatile Purification Methods 3.2.4 Electrophoretic Methods are Used for Preparation and Analysis 3.2.5 Purification of Specific Proteins Involves Combination of Different Procedures Summary 1. Protein solubility is not a fixed quantity for a given protein. Rather, it is a function of many variables. Two of these are pH and salt concentration. Proteins show a minimum solubility at their isoelectric point. Frequently, proteins require the addition of a small amount of salt to become soluble, but excessive amounts of salt lead to protein precipitation. 2. There are several methods for determination of molecular weight. These include sedimentation analysis and gel-exclusion chromatography. Sedimentation analysis may be used in two different ways: (1) by independently determining the sedimentation and diffusion rates and combining this information to calculate a molecular weight and (2) by equilibrium ultracentrifugation. Gel-exclusion chromatography uses cross-linked polydextrans and relates molecular weight to the rate of migration through a column. 3. Electrophoretic methods are used in various ways to characterize protein mixtures and purified proteins. The high resolution attainable by electrophoresis makes it ideal for determining the number of proteins in a mixture as well as their approximate size 4. Methods of protein purification include differential centrifugation, differential precipitation with (NH4)2SO4, gel-exclusion chromatography, different electrophoretic mobility, and differential affinities for column matrice containing different functional groups. Column procedures are particularly versatile because of the large number of functional groups that can be used to bind proteins in different ways and because of the variety of conditions for differential column elution. Chapter 4 Primary Structure of Proteins: The Building Blocks of Proteins----Amino Acids, Peptides, and Polypeptides Key-points: From our presentation you will learn the following: 1. Certain acidic and basic properties are common to all amino acids found in proteins except for the amino acid proline. 2. Side chains give amino acids their individuality. These side chains serve a variety of structural and functional roles. 3. The alpha-carboxyl group of one amino acid can react with the alpha-amino group of another amino acid to form a dipeptide. 4. Many amino acids, reacting in a similar way, can become linked to form a linear polypeptide chain. 5. The Amino acid sequence in a polypeptide can be determined by a process of partial breakdown into manageable fragments, followed by stepwise analysis proceeding from one end of the chain to the other. 6. Polypeptide chains with a prespecified sequence can be synthesized by well-established chemical methods. ,,,,,,,,,,,,,,,,,,,,,, 4.1 Amino acids Figure 1 Amino acid anatomy Table 1 Structure of the 20 amino acids found in protein 4.1.1 Amino acids have both acid and base properties Fig 4.3 Equilibrium between charged and uncharged forms of amino acid side chains Table 4.2 Values of pK for the ionizable groups of the 20 amino acids commonly found in proteins 4.1.2 Aromatic amino acid absorb light in the near- ultraviolet Fig 4.4 Ultraviolet absorption spectra of Trp, Tyr, and Phe at pH 6. 4.1.3 All amino acids except glycine show asymetry Fig 4.5 The covalent structure of alanine, showing the three-dimensional structure of the L- and D- stereoisomeric forms 4.2 Peptides and polypeptides Fig 4.6 Formation of a dipeptide from two amino acids Fig 4.7 A polypeptide chain, with the backbone shown in color and the amino acid side chains in outline 4.3 Determination of amino acid composition of proteins To determine a protein‘s amino acid composition, it is necessary to (1) break down the polypeptide chain into its constituent amino acids, (2) separate the resulting free amino acids according to type, and (3) measure the quantities of each amino acid. Tab 4.3 Amino acid content of protein (in precent) 4.4 Determination of amino acid sequence of proteins The amino acid sequence of the first protein (insulin) was determined in 1953 by Sanger‘s laboratory. The importance of knowledge of the amino acid sequence of the proteins shows in a number of ways: It permits comparisons to be made between normal and mutant proteins; It permits comparisons to be made between comparable proteins in different species and thereby has been instrumental in positioning different organisms on the evolutionary tree; Final and most important, it is a vital piece of information for determining the three-dimensional structure of the protein. The steps to determine the amino acid sequence of the protein(Fig 4.13): Purification of the protein Cleavage of all disulfide bonds Determination of the terminal amino acid residues Specific cleavage of the polypeptide chain into small fragments in at least two different ways Independent separation and sequence determination of peptides produced by the different cleavage methods Reassembly of the individual peptides with appropriate overlaps to determine the overall sequence Figure 4.14 Disulfide cleavage reaction Figure 4.15 Polypeptide chain end-group analysis Figure 4.16 The Dansyl chloride method for N-terminal amino acid determination Figure 4.17 The cleavage of polypeptide chains at methionine residual by cyanogen bromide Figure 4.18 The Edman degradation for polypeptide sequence determination 4.5 Chemical synthesis of peptides and polypeptides Chapter 5 The Three-Dimensional Structures of Proteins Proteins adopt the most stable folded structure, this is a function of the way in which the individual amino acid residues interact with one another Two famous scientists, Pauling and Corey made a great progress in deduced protein structure, using information from various sources: They knew a little about the structure of peptides from small-molecule crystallography, which indicated that the peptide bond was planar and gave accurate bond lengths and angles They were already aware of the importance of hydrogen bonds in determining the orientation of amino acids, peptides and even water in simple crystals They made shrewd guesses about the interpretation of a few spacings in the diffraction patterns of certain fibrous proteins Putting all of this information together, they experimented with molecular models until they could produce structure in reasonable agreement with all the available facts. Kendrew and Perutz: myoglobin and hemoglobin Enormous advances have been made in protein chemistry and in computer technology as well: The final folding agreements of proteins may some day be predictable from the amino acid sequences of the polypeptide sequences 5.1 The information for folding is contained in the primary structure The conformation of a native or highly organized proteins reflects a delicate balance among a variety of interaction forces, both within the folded protein‘s interior and with surrounding solvent. Example: Figure 5.1 Schematic representation of an experiment to demonstrate that the information for folding into a biologically active conformation in contained in the protein’s amino acid sequence. Chaperons: a special class of proteins that appear to catalyze the folding process of polypeptide chain into a native conformation 5.2 The Ramachadran Plot predicts sterically permissible strucutre Figure 5.2 Basic dimensions of (a) the peptide and (b) the dipeptide Figure 5.4 Ramachandran plot Figure 5.5 Ramachandran plot(2) In summary, it can be seen that owing to the basic geometric properties of the polypeptide chain, its sterically allowed conformations are severely restricted by the occurrence of unfavorable steric interactions between various atomic groups. 5.3 Protein folding reveals a hierarchy of structural organization Anfinsen‘s experiment: protein folding is a spontaneous process. In actuality, newly synthesized polypeptide chains typicaly fold in seconds. This means that protein folding must be a highly directed and cooperative process. Although much remains to be learned about the details of the process, its speed and its facility suggest the existence of a sequential set of folding intermediates, each being more highly organized than the one before it. Figure 5.6 Possible successive steps in the protein folding process. The forces that stabilize protein act in concert with related energetic and geometric factors to yield successively large and more complex protein structural arrangement. Figure 5.7 Herarchies of protein structure To begin with, steric interactions restrict accessible conformations and reflect feature of protein‘s amino acid sequence, or Primary structure. The requirement for hydrogen-bond preservation in the folded structure result in cooperative formation of regular structural regions in proteins. This situation arises principally because of the regular repeating geometry of the hydrogen-bonding groups of the polypeptide backbone an leads to the formation of regular hydrogen-bonded Secondary structures. Association between elements of secondary structure in turn results in the formation of Strucutral Domains, whose properties are determined both by chiral properties of the polypeptide chain and packing requirements that effectively minimize the molecule‘s hydrophobic surface area. Further association of domains results in the formation of the protein‘s Tertiary Structure, or over-all spatial arrangement of the polypeptide chain in three dimensions. Likewise, fully folded protein subunits can pack together to form Quaternary Structure, which can serve a structural role or provide a structural basis for modification of the protein‘s functional properties 5.4 Two secondary structure are found in most proteins A major driving force in folding is the necessity to minimize the extent of exposure b the hydrophobic group to solvent. This consideration involves a sacrifice of the favourable hydrogen-bonded interactions between the unfolded polypetide backbone and water. To preserve a favorable energy balance of folding, the backbone polypeptide groups must take part in alternative hydrogen-bonded interactions between themselves in the protein‘s folded state. 5.4.1 The , helix Characterizing Parameters of the , helix: (1) each residue‘s carbonyl group forms a hydrogen bond with the amide NH group of the residue four amino acids farther along the polypeptide chain; (2) all residue in an , helix have nearly identical conformations, averaging ,=-45º to -50º and ,=-60º, so they lead to a regular strucuture in which each 360º of helical turn incorporate approximately 3.6 amino acid residues and rise 5.6Å along the helix axis direction. (3) The advance per amino acid residue along the helix axis is 1.5Å. Figure 5.8 Three ways of projecting the , helix. An important property stemming from the conformational regularity of the , helix, which applied to other secondary structures as well, is cooperativity in folding. 5.4.2 The , Sheet Figure 5.9 The antiparallel , sheet , sheets occur in two different arrangements. In the first of these, the chains are arranged with the same N-to-C polypeptide sense to produce a parallele , sheet. Alternatively, the chains can be aligned with opposite N-to-C sense to produce an antiparallel , sheet. Figure 5.10 Two forms of the , sheet structure 5.5 Pauling and Corey provided the foundation for understanding of fibrous protein structure Linus Pauling and Robert Corey examined the structure of crystals formed by amino acids and short peptides and formed two rules that describe the ways in which amino acids and peptides interact with one another to form noncovalently bonded crystalline structures. These rules laid the foundations for our understanding of how amino acids in protein polypeptide chain interact with one another: st 1 rule: was that the peptidyl C-N linkage and the four atoms to which the C and the N atoms are directly linked always forms a planar structure;(which indicates the only flexibility in the polypeptide backbone arises from rotation about the carbon that joins adjacent peptide planar groups) nd 2 rule: was that peptidyl carbonyl and amino groups always form the maximum number of hydrogen bonds. Taken together, these two rules drastically reduce the number of possible conformations available to the polypeptide chain. 5.6 Collagan forms a unique triple-stranded structure 5.7 In globular proteins, secondary structure element are connected in simple motifs Figure 5.17 The structure of lysozyme--- the first enzyme whose three-dimensional structure was determined, the120-residue protein This protein has local regions of ordered ,-helical and antiparallel ,-sheet secondary structure. In addition it has several additional regions of single-stranded loops with a less regular conformation. At the most elementary level of structural analysis, it was found that simple combinations of a few secondary structure elements with specific geometric arrangements are used again and again in different protein structure. Three of these structural motifs that are used most frequently: (1) The helix-loop-helix (Figure 5.18) (2) The hairpin , motif (Figure 5.19, 5.20, 5.21) (3) The ,-,-, motif (Figure 5.22) 5.8 The domain is the basic unit of tertiary structure A domain constitutes a stable unit of tertiary structure; it usually contains a combination of two or more covalently linked structural motifs. Some proteins contains a single domain. Others contain two or more domains held together by covalent linkages or noncovalent linkages. While it is clear that there is an enormous variety of domains, it is remarkable how many times we find strikingly similar domains in different proteins and how often it is possible to gain an immediate qualitative understanding of the features that account for a protein‘s stability and function once the structure has been determined. No protein is found as a single-layer structure. This is because it requires at least two layers to bury the hydrophobic core resulting from the hydrophobic amino acid side chains that are inevitably found in all proteins. The three kinds of domains: 5.8.1 The helix-loop-helix motif is the basic component found in , domain structures Two commonly found domains of this type: (1) four-helix bundle (Figure 5.23 & 24) (2) globin fold (Figure 5.25) 5.8.2 ,/, domains exploit the ,-,-, motif (Figure 5.26) The most frequent and most regular of the domain structures are the ,/, domains, which consist of a central parallel or mixed , sheet surrounded by , helices.Most domains of this type make extensive use of the ,-,-, motif. (e.g. glycolytic enzymes, translocating proteins, etc.) 5.8.3 Antiparalle , domains show a great variety of topologies (Figure 5.27~29) 5.8.4 Some proteins or domains require additional features to account for their stability (Figure 5.30) In addition to the packing of elements of protein secondary structure, which is a dominant feature in most proteins, there are cases, especially among the samllest structures, where the geometry and presence of disulfide bonds or nonpeptidyl groups are a dominant factor. Special structural features that account for the stability of membrane-binding proteins and DNA-binding proteins will be discussed in details later. 5.8.5 Many proteins contain more than one domain Within a single subunit, contiguous portions of the polypeptide chain often fold into more than one domain. Sometimes the domains within a protein are very different from one another (Figure 5.31), but often resemble each other very closely (Figure 5.32). Figure 5.33~34 5.9 Quaternary structure depends on the interaction of two or more proteins or protein subunits Quaternary structure: The higher-order organization of globular subunits to form a functional aggregate. Types: (1) subunits that resemble to form the quaternary structure are very different in structure (2) a commonly observed pattern of quaternary structure is typified by molecular aggregate composed of multiple copies of one or more different kinds of subunits. 5.10 Predicting protein structure from protein prmary structure (omitted) 5.11 Methods for determing protein conformation 5.11.1 X-ray diffraction analysis of fibrous proteins 5.11.2 X-ray diffraction analysis of proteins crystals 5.11.3 Nuclear magnetic resonance (NMR) complements X-ray crystallography 5.11.4 Optical Rotatory Dispersion (ORD) and Circular Dichroism (CD) Chapter 6 Protein Structure Prediction Outlines: Protein structure prediction is becoming urgent because of the increased discrepancy between the number of known protein sequences and the number of experimentally-determined structure. In this chapter, we will discuss (1) the principles of protein structure as they related to the prediction problem; (2) the approaches of protein structure prediction; and (3) some examples. 6.1 Introduction The central dogma motivating prediction is that the three-dimensional structure of a protein is determined by its sequence and its environment without the obligatory role of extrinsic factors. This hypothesis comes mainly from the classic study of re-naturation of ribonuclease, which is conformed by many experimental results. However, it has been challenged since then, such as that chaperons and disulfide interchange enzymes have been identified as assisting the folding process. Some other experiments support this hypothesis, i.e. these molecules just help but not determine the final natural state of proteins. There has been sufficient success of predictions to justify the use of the central dogma as a working hypothesis. Prediction is becoming a pressing problem for many biologists as the discrepany continues to increase between the number of known protein sequences and the number of experimentally-determined structure (Fig. 6.1) 6.2 Principles of protein structure (Factors deteriming protein structure) 6.2.1 Dominant effects in protein folding In theory, molecular dynamics simulation in solvent with accurate potentials and run over sufficient time would model the folding of a protein. Since this is not feasible at present, it is instructive to describe the individual effects that govern the protein/solvent system. 1. Net protein stability The diverse chemical properties of the protein main chain and side chains (Figure) give rise to an interplay of non-covalent and entropic effects that determine the structure of the molecule. Most globular water soluble proteins have only marginal stability at their physiological conditions: the change in Gibbs free energy from the unfolded to folded state typically is between –5 to –20 kcal mol-1. Understanding and quantifying the thermodynamic effects remains a chanllenge. 2. The hydrophobic effect It is widely regarded that protein folding is driven by the hydrophobic effect. This describes the energetic preference for non-polar atoms, such as hydrocarbons, to associate and reduce their contact with water. At room temperature, the effect is mainly entropic. Experimental measures of the magnitude of hydrophobicity for different side chains come from partition experiments in which the concentrations of compounds modelling side chains are measured in a medium representing the protein core and in water. The relationship between the hydrophobic effect and the accessible surface area (ASA) of the solute has dominated many aspects of protein modelling. ASA is defined as the locus of the centre of a water probe as its rolls around the surface of a molecule (Fig). Molecular surface is the sum of the area of the solute atoms in contact with this water probe (contact surface) and the re-entrant surfaces of the water probe. There is an approximate linear relationship between hydrophobicity and the ASA of all non-polar atoms in side chains in their extended conformation. 3. Atomic packing The net effect of attractive and repulsive van der Waals interactions between atoms is to favor close atomic packing. Thus to a first approximation the protein core resembles the solid state. Surface residues and most atoms of the chain in the unfolded state are less ordered and resemble in part the liquid state. Thus for residues that are in the protein core, folding leads to a liquid,solid transition. This transition is primarily enthalpic. 4. Conformational entropy the formation of the folded structure restricts the dihedral conformational space samples by the main chain and the buried side chain. This freezing of rotamers is entropically unfavorable. 5.Electrostatic effects---ion pairs and hydrogen bonds The net effects of hydrophobicity, close packing, and conformational entropy would probably lead to a compact protein that lacks a specific architecture. The specificity for the tertiary structure could be considered as residing in the location of the hydrogen bonding and the ion pairing groups. Electrostatic effects in the protein/solvent system are complex. An individual fully charged (or just apolar atom) extending from the protein surface into water will be surrounded by a solvation shell of water molecules. Transfering this charged atom the protein core is energetically very unfavorable due to removing the solvation shell. Thus, isolated charges are very rarely observed buried within proteins. The formation of protein-protein electrostatic interactions must compete with the charges interacting with water and thus a charge interaction will be far less favorable energetically compared to the in vacuo effect. Despite the disadvantageous effect of partial desolvation, ion pairs on the surface tend to stabilize a protein and on average one-third of charged residues in a protein are involved in salt bridges. However there is an adverse effect of burying an ion pair in a low dielectric environment with only about 20% of such pairs being fully buried. There is a competition between protein-protein and protein-solvent hydrogen bonds. Although hydrogen bonds abound in proteins both forming secondary structures and involving side chain/main chain and side chain/side chain interactions, it remains unclear whether hydrogen bonds, particularly if buried, actually stabilize a protein. The formation of ,-helices and ,-sheets is probably the consequence that the periodic hydrogen bonding provides the best method of arranging complementary main chain amide and carboxyl groups within a hydrophobic core. 6. Disulfide bridges The common view is that disulfide cross-links stabilize the folded state by entropically restricting the degrees of freedom of the unfolded state compared to the same chain without cross-links. For a single link, the stability increases with the length of the link but for multiple bridges there are complex effects. Typically a link will yield a few kcal mol-1 of stability. There are small proteins whose stability is considered to be enhanced by the entropic effect of multiple disulfide bridges. 6.2.2 Analyses of Protein Structure Many workers primarily follow a knowledge-based (i.e. empirical) approach to structure prediction. However, as more protein structures were determined to high resolution, analyses were increasingly able to identify principles of protein architecture. These principles then form the basis for predictive algorithms. Some features that are particularly relevant to prediction are highlighted below: 1. Residue conformation (1) The main chain backbone torsion angles adopt allowed states conventionally represented as a Ramachandran (,, ,) plot. Gly adopts a larger and Pro a smaller region of allowed (,, ,) space. (2) Proline is the only residue that adopts a cis peptide conformation with a relatively high probility (3) Side chains adopt distinct conformations that are dependent on backbone conformation. These conformations are conviently represented as rotamer libraries 2. Periodic secondary structure (1) ,-helices can be curved or bent due to interactions with solvent, the presence of proline, or an ,-aneurism (2) There are differentpreferences for residues to occur in the middle of an ,-helix, and at the three N-terminal residues (N-cap), just before the N-cap, at the C-cap, and just after the C-cap. (3) Nearly all ,-sheets have a right-handed twist along the strand direction and consequently a left-handed twist between strands. (4) A common distortion to the ,-sheet is the ,-bulge. (5) Certain residues preferentially occur within ,-strands. (6) Right-handed 3.10 helices are relatively common (about 4% of residues). These helices can occur independently (typically less than six residues) or can form a terminal few residues of an ,– helix. (7) Left-handed polyproline II helices are relatively common (about 4% of residues) but their recognition was delayed due to the absence of periodic hydrogen bonds. These helices tend to be less than six residues long. 3. Non-periodic secondary structure (a) A ,-turn refers four residues that achieve a 180: chain reversal. There are preferred sequence patterns for the different conformational families of ,-turns (b) ,,,,,,,,,,, Chapter 7 Protein Folding and Unfolding C. B. Anfinsen discovered that the small proteins ribonuclease A and staphylococcal nuclease could be reversibly denatured. On removal of a chemical denaturant, such as urea, they spontaneously refold to their native structures after denaturation. Similarly, they spontaneously refold on cooling after thermal denaturation. Not only the amino acid sequences of these proteins encode their final folded structures, they also encode the information on how to get to the structures. But there are proteins that will not renature after being denatured, sometimes because they have been processed after biosynthesis. In some cases, larger or multimeric proteins do require assistance to fold, which is provided by molecular chaperones. The kinetics of folding and unfolding appear to be a very complex process, but this process follows very simple rate laws, governed by a few basic principles. 7.1 Kinetic analysis of complex reactions To determine the mechanism and pathway of unfolding and refolding, the intermediates that define and direct the pathway must be identified, but these are usually unstable thermodynamically. They might be detectable as kinetic intermediates, but only if they occur on the pathway before the rate-limiting step and if their free energies are comparable to or lower than that of the initial state. No other kinetic intermediates are populated to substantial levels, even transiently. With a simple one-step reaction followed as a function of time, t, there is expected to be a single kinetic phase, characterized by a single rate constant k: Fraction folded conformation = 1-exp(-kt) (7.1) More complex kinetic behavior would be observed either if there were rate-limiting steps in the reaction or if the starting material were heterogeneous, with different populations having different rates of reaction. Discriminating between these possibilities with a multiphase reaction is not always straightforward. It is too often assumed that one kinetic phase of a folding reaction represents formation of an obligatory intermediate I and that a second phase represents its conversion to another intermediate or to N: k -1 U•I • N (7.2) k k 12 if this were the case, there would be a lag period in the appearance of N -1of approximate magnitude (k + k), during which the steady-state 12 concentration of I would be generated. This effect is cumulative with additional steps. Therefore, the magnitude of the lag period in formation of the final folded conformation should be correspondingly longer with an increasing number of obligatory, sequential intermediates along a pathway. Most kinetic complexities of protein folding arise from heterogeneity of the unfolded state. Because of the great conformational heterogeneity of the unfolded state, protein folding is a special kinetic phenomenon in which every molecule of a typical population is likely to have a unique conformation at every instant. For example, 1 mg of a protein with a molecular weight 416of 10 comprises 0.1 ,mol, or 6，10 molecules, whereas many more conformations are likely when it is unfolded. How is this conformational heterogeneity apparent in the kinetics of refolding? Does each molecule refold at its own rate, determined by its conformation at time zero, or do molecules somehow fold by a common mechanism at a common rate? If each molecule does not fold uniquely, how do different molecules manage to follow the same rate-limiting step? It is unrealistic to expect to elucidate all the details of a complex reaction like protein folding. Although it occurs much more rapidly (on second to minute time scale) than expected for a random search, this time is long enough for each molecule to undergo perhaps some 111310~10 conformational changes. Because each molecule of a substantial population starts out with a different conformation, it might be feasible to determine at what stage different molecules start to follow the same pathway. At best it may be possible only to characterize the slowest transitions and the conformations and energetics of the most stable intermediates, to identify the overall rate-limiting step, and to characterize the transition state. 7.2 Kinetics of unfolding Unfolding of proteins is almost universally observed to be an all-or-none process, with little or no partial unfolding preceding complete unfolding. When a native, covalently homogeneous protein is placed in unfolding conditions at time zero, unfolding almost always occurs with a single kinetic phase and a single rate constant. There is no lag period, and all probes of unfolding give the same rate constant. Therefore there is a single rate-limiting step in unfolding, and all folded molecules have the same probability of unfolding. Exceptions generally result from exceptions to the usual homogeneous nature of the folded state. The rate of unfolding usually changes uniformly with variation of the unfolding conditions. In particular, logarithmic plots of unfolding rates versus denaturant concentration or temperature are generally linear, suggesting that the mechanism of unfolding is not changing. There appears to be a single transition state for unfolding under these conditions. 7.3 Kinetics of refolding Kinetic complexities are encountered almost universally in protein refolding. These complexities usually result from conformation heterogeneity of the unfolded state, with slow- and fast-refolding molecules: slow fast U•U•N (7.3) SF In virtually all characterized cases, the heterogeneity arises from cis- trans isomerization of peptide bonds preceding Pro residues. 1. Peptide bond isomerization Cis peptide bonds are often found in folded proteins, but almost only when the next residue is pro. A peptide bond is usually cis or trans in essentially all the folded molecules because the folded conformation generally favors one over the other. When a protein is unfolded, however, the constraints favoring one form over the other are released, and an equilibrium between cis and trans isomers is attained at each peptide bond. When the protein is refolded, a fraction of the molecules, U, have all the necessary peptide bonds as the correct isomer whereas F the others, U, have one or more s an incorrect isomer. S Cis-trans isomerization of Pro peptide bonds is intrinsically slow(Sec.5.2.4.b). When the rate of refolding of the U molecules is F faster than cis-trans isomerization, U and Umolecules have different FS rates of refolding(Eq.7.21) If all the peptide bonds must be of the correct isomer for refolding to occur, the greater the number of Pro residues the greater the fraction of U molecules and the slower their refolding. The S actual situation is more complex, however, because some proteins can refold to a native like conformation with an incorrect isomer of one or more peptide bonds. Also, the rates of isomerization can be either increased or decreased by the conformation of the protein. For example, two of the four Pro resudues in bovine ribonuclease A have cis peptide bonds in the folded conformation. Unfolded ribonuclease A (with the four disulfied bonds intact) refolds in three different kinetic phases, corresponding to at least three different unfolded species. One accounts for 15% of the molecules and refolds within less than a second under optimal conditions; it is thought to have all correct peptide bond isomers. The remaining 85% of the molecules are thought to have one or more nonnative peptide bond isomers that must slow refolding. A second kinetic species represent 65% of the molecules and refolds on a time scale of seconds. Under conditions strongly favoring folding, this species folds more rapidly into a native-like formation, retaining the incorrect peptide bond isomer. The remaining 20% of the molecules make up the third kinetic species and refold even more slowly. The second and third kinetic species are believed to result from trans isomers predominating in the unfolding. 2. Refolding in the absence of slow peptide bond isomerization In a population of unfolded molecules with the same cis-trans isomers as the native state, the refolded protein generally appears with a single rate constant and without a significant lag period. The absence of an observable lag period indicates that there is a single rate limiting step in refolding and that all preceding and subsequent steps are more rapid. Consequently, refolding can be simplified to three stages (Fig. 7.29): (1) the nature of the unfolded protein under refolding conditions, the ―prefolded‖ conformation; (2) the nature of the rate limiting step and the overall transition state for folding; and (3) the nature of the folded conformation under refolding conditions, especially its flexibility. Considering the conformational heterogeneity of the unfolded state (but excluding intrinsically slow isomerizations), it is noteworthy that all the molecules with the same covalent structure are usually observed to fold with the same rate constant. A single rate constant is consistent with all the molecules folding by the same rate-determining step. The folding of many conformationally heterogeneous molecules by the same rate-limiting step requires that there be a rapid conformational equilibration prior to the rate-limiting step (Fig.7.29C). That this occurs is also indicated by the general observation that the rate of refolding depends only on the final folding conditions, not the initial unfolding conditions. Proteins unfolded in different ways generally have different average physical properties. Nevertheless, they refold at indistinguishable rates under the same final folding conditions. The rate of folding is determined not by the nature of the initial unfolded protein but by the properties it rapidly adopts when placed under the final folding conditions. How do all unfolded molecules equilibrates rapidly prior to refolding if sampling of all conformations by a random coil requires such a long period of time? The answer undoubtedly is that an unfolded protein under refolding conditions does not behave as a random coil but adopts a limited set of energetically favored nonrandom conformations. In this way, all the molecules converge to follow a common subsequent pathway and have the same rate-limiting step. This convergence is in contrast to the proposal that each protein molecule folds by a unique pathway. The rate of direct refolding generally varies with temperature in a complex manner, giving a nonlinear Arrhenius plot. At low temperatures, the rate of refolding increases with temperature, as do most chemical reactions. The increase in rate diminishes, however, and the rate reaches a maximum and then decreases dramatically at high temperatures. This temperature dependence is unusual for chemical reactions, but it might be expected for a complex reaction like protein folding that is dependent on the presence of metastable, partially folded intermediates. Such metastable intermediates would be destabilized at high temperature, and the rate of refolding would decrease accordingly. 3. The prefolded state The prefolded state is the unfolded protein under refolding conditions, prior to the rate-limiting step and complete refolding. The prefolded state is intrinsically unstable and is populated only transiently. Nevertheless, a variety of evidence indicates that it has considerable nonrandom conformation in many proteins. The nature of the prefolded state is being investigated very actively, using its spectral properties, its susceptibility to proteases, protection from exchange of its labile hydrogens, and the effects of mutagenesis. 4. The transition state for folding 7.4 Folding pathways Elucidating the mechanism of protein folding requires characterization of the initial, intermediate, and final conformational states, plus determination of the steps by which they are interconverted. The kinetic roles of the various states can be determined most readily if there is some means of control over the rates and equilibria of the various steps. This control would also make it possible to ensure that unstable intermediates accumulate to substantial levels, at least transiently. Ideally, the unstable intermediates would be trapped in a stable form so that they could be characterized. To control the rate of formation and breakage of hydrogen bonds would be almost ideal because every protein structure includes hydrogen bonds. During folding, protein molecules with 1, 2,3,….intramoleular hydrogen bonds might accumulate kinetically, if they could be trapped and identified, a pathway could be defined in terms of hydrogen bonds. Unfortunately, it is not possible to trapped, due to the reduction-oxidation nature of the covalent disulfide interaction between thiol groups. 1. Trapping intermediates with disulfides some proteins that contain disulfide bonds in their folded conformations require these disulfide for stability of their folded conformation. In this case, the reduced protein is unfolded, even in the absence of denaturants, and folding and disulfide bond formation are coupled. Protein species with different numbers of disulfide bonds that accumulate during unfolding and refolding can be trapped and separated and their disulfide bonds identified. The kinetic roles of the intermediate can often be determined unambiguously due to the ability to control the kinetics and thermodynamics of the disulfide interaction. Under approriate condition, the disulfide interaction can be very dynamic, with disulfides being formed, broken, and rearrangd on time scales as short as 10-5s. the rates of the intermolecular steps in disulfide formation reflect the protein conformational transitions involved. The approach is useful only with proteins that unfold when their disulfides are broken; unfolding and refolding of the protein consequently can be controlled by varying jus the intrinsic disulfide stability. There is no need to use denaturants, and the strengths of all other types of interactions that stabilize protein are not affected. Although only the disulfide bonds are trapped, the conformations that direct disulfide bond formation are effectively trapped also. The stabilities of protein disulfides and of the conformations that specify them are linked functions. It is thus a thermodynamic requirement that whatever conformation stabilize a particular disulfide bond must be stabilized to the same extent by the presence of that disulfide. Therefore, the conformational basis of folding should be evident from the conformation of the trapped intermediates as long as the conformations are not affected by the trapping procedure. 2. Disulfide folding pathway of BPTI Examples: p 318~ Figure 7.53 The disulfide folding pathway of BPTI (bovine pancreatic trypsin inhibitor) 7.5 Folding of large proteins Large proteins are composed of multiple structural domains, multiple subunits, or both. Individual domains can often be excised proteolytically from a protein, or the corresponding fragment can be produced by protein engineering. In many cases, the isolated domains are as stable as when they are in the intact protein, and they are independent structural units in the intact protein. The independent domains unfolded and refold like single-domain protein, which can lead to complex unfolding curves for a protein when its domains unfold under different conditions. There can also be varying degrees of interaction between the domains when they are part of the same molecule. If these interactions are mutually stabilizing, the isolated domains are corresponding less stable. In extreme cases, domains can be so independent as to become a single cooperative unit. When the isolated domains are stable, folding of an intact multidomain protein appears to occur by folding of individual domains, followed by their association. Somewhat surprisingly, the domains linked by a polypeptide chain often fold more slowly than when they are isolated. The various segments of a polypeptide chain seem to interfere with the folding of each other. Association of the folded chains is often the slowest step in the overall folding process, either because the domains are not folded entirely correctly or because the small adjustments required for their interaction are energetically unfavorable. When association of folded domains is slow, an intermediate state accumulates during folding in which the individual domains are folded but unpaired. These domians apparently can interact with the complementary domains of other molecules, which often leads to indefinite aggregation of the protein and to its precipitation. For this reason, productive folding of large proteins usually must be carried out at very low protein concentrations. The folding of oligomeric proteins is subject to similar considerations because their subunits often consist of multiple domains. With oligomers, however, specific interactions between molecules are necessary. the monomers generally fold to nearly their final conformations before any association steps occur, specific association presumably requires a folded conformation to provide the interaction site. Nevertheless, further folding generally occurs after association. The rate-limiting step in regenerating a native oligomeric protein can be either intramolecular folding or association of two particles. Which is rate-limiting often depends on the protein concentrations. No scheme for folding and assembly is general to all oligomeric proteins. This should not be surprising in view of the many different quaternary structures that are encountered in proteins (Fig.6.24). But even homologous proteins with essentially the same quaternary structure can use apparently different assemble mechanisms. Association of folded monomers would seem to be a straightforward process, but it often observed to be relatively slow, with association 35-1-1constants of only 10-10 m s. The final adjustments of the structure upon association seem to involve a significant energetic barrier. 7.6 Biosynthetic folding Proteins are synthesized on ribosomes in vivo as linear polypeptide chains, but they rapidly fold to their final conformation either during or after biosynthesis. Very little is known directly about how or when this happens in vivo. 7.1.1 Basic methods The folding of proteins is usually studied in vitro by first denaturing them in solution of urea, guanidinium chloride, or acid and then diluting the denaturant. Stopped-flow methods are the most convenient, because they cover a useful time range and are ideal for mixing two reagents. Fluorimetry, following the change in tryptophan fluorescence in the near ultravilolet, is used because of its sensitivity. This technique is generally used to monitor tertiary interactions because the fluorescence yield and emission wavelength of tryptophan are sensitive to its environment. Stopped-flow circular dichroism is useful because it can detect changes in secondary structure in the far ultraviolet. Helixes form in a few hundred nanoseconds and , turns in a few microseconds 6-1in model peptides. Short loops in proteins form with an upper limit of about 10 s. Thus, a lower limit for the initial collapse of a denatured protein is about 1s. , Conventional rapid mixing methods are limited to a time scale of milliseconds or greater, but specialized continuous-flow apparatus has been used for tens of microseconds. Relaxation methods or flash photolysis are necessary for investigating faster reactions. …… 7.1.2 Multiple phases and cis-peptidyl-prolyl bonds 1. Effects of peptidyl-proline isomerization on kinetics Refolding is generally found to proceed by a series of exponential phases. Many of these exponentials are a consequence of cis-trans isomerization about peptidyl-prolyl bonds. The equilibrium constant for the normal peptide bond in 34proteins favors the trans conformation by a factor of 10-10 or so. The peptidyl- prolyl bond is an exception that has some 2-20% of cis isomer in model peptides. Further, it is often found as the cis isomer in native structure.The interconvension of cis to trans in solution is quite slow, having half-lives of 10-100s at room temperature and neutral pH. This has two important consequences. First, a protein that has several proline all in the trans conformation in the native structure will equilibrate when denatured to give a mixture of cis and trans forms. Chapter 8 Stabilizing Protein Function One of the great unresolved problems of science is the prediction of the three-dimensional structure of a protein from its amino acid sequence: ―folding problem‖. An even more elusive goal is the prediction of the catalytic activity of an enzyme from its amino acid sequence. Why so important? 1. the acquisition of sequence data by DNA sequencing is relatively quick, and vast quantities of data have become available through international efforts such as the Human Genome Project and other genome sequencing projects. The acquisition of three-dimensional data is still slow and is limited to proteins that either crystallize in a suitable form or are sufficiently small and soluble to be solved by NMR in solution. Algorithms are thus required to translate the linear information into spatial information; 2. we are now able to synthesize proteins by way of their genes, and so the production of new enzymes with specified catalytic activities is a challenging prospect. Producing such an new enzyme requires five underpinning and interrelated abilities: (1) the ability to predict the most stable fold of a particular sequence; (2) the ability to design a novel fold; (3) the ability to predict whether the desired fold is kinetically accessible; (4) the ability to design the precise features for specific binding in the fold; and (5) the ability to design the precise orientation of groups in the protein for efficient catalytic function. 8.1 Understanding Protein Stability 8.1.1 Protein Stability and Its Measurement 1. Introduction Protein stability is a very important area of study within biotechnology, Because: (1) For enzymes, although enzymes are protein molecules that act as extremely efficient catalysts, the usefulness of enzymes and proteins as analytical tools and as industrial catalysts is often limited by their requirements for ―mild‖ storage and reaction conditions. This is because many emzymes lose their catalytic abilities over time, that is, they have poor operational or long- term stability; (2) Stability is an issue also in the development and use of protein- based analytical or sensor devices, and it can be of literally vital importance in protein pharmaceuticals or therapeutics, where deterioration of protein preparations over extended storage periods can be a serious drawback; (3) Interesting in protein stability will likely grow due to the increasing use of recombinant therapeutic proteins, the advent of protein engineering and recent strides in understanding protein folding. 2. Protein Structure(see those have already been discussed) …….. It is an axiom in biology that structure relates to function. Integrity of the three-dimensional tertiary (or quaternary) structure is essential for the correct functioning of the protein, e.g. catalysis of a reaction by an enzyme, or antigen binding by an antibody. Loss of quaternary or tertiary structure leads to loss of function. Adverse conditions of temperature, pH or solvent, or high concentration of urea or quanidinium hydrochloride, can bring about this loss of function. Heavy metals, certain organic chemicals or chelating agents can act similarly in some cases. 3. Definition and measurement of protein stability The term ―stability‖ refers to a protein‘s resistance to adverse influences such as heat or denaturants, that is, to the persistence of its molecular integrity or biological function in the face of high temperatures or other deleterious influences. A perfectly folded, fully functional monomeric protein can lose its biological activity in vitro by unfolding of its tertinary tructure to a disordered polypeptide, in which key residues are no longer aligned closely enough for continued participation in functional or structural-stabilizing interactions. Such unfolding is termed denaturation. It is usually cooperative and may be reversible if the denaturing influence is removed, since the polypeptide chain has not undergone any chemical changes. A protein is also subjected to chemical changes which lead to an irreversible loss of activity or inactivation, particularly following unfolding. An unfolded, extended polypeptide will be much more prone to proteolysis than a tightly-packed, globular protein. Unfolding may result in the loss of a functionally-essential cofactor from a holoprotein, such that biological activity will not be regained even if the unfolding can be reversed to yield the corresponding apoprotein. Unfolded polypeptide chains may aggregate to form an inactive, insoluble mass while an individual chain attempting to refold may enter an incorrect, kinetically-trapped conformation from which it cannot emerge. (A living cell may be able to prevent these events, cells contain a number of so-called chaperone proteins which assist the folding on newly-synthesized proteins in vivo. Chaperones can also act in vitro to prevent aggregate and assist folding. At least one such protein has been shown to reactivate an aggregated enzyme.) These different molecular phenomena give rise to two distinct definitions of in vitro protein stability: Thermodynamic (or conformational) stability: it concerns the resistance of the folded protein conformation to denaturation (i.e. its Gibbs, or free, energy of unfolding); (N,,U ) Long-term stability: it measures the resistance to irreversible inactivation (i.e. persistence of biological activity.) (U,,I) Both types can be represented in a single scheme: Kk N,,U,,I K: the equilibrium constant for the reversible reaction k: the rate constant for the irreversible reaction. 4. Folding stability Folding (conformational)stability is usually measured by optical techniques (such as UV spectrophotometry, fluorescence or circular dichroism) or by urea gradient gel electrophoresis. Recent reports describe the analysis of thermal denaturation by free solution capillary electrophoresis and temperature gradient gel electrophoresis. These methods are sensitive to changes in protein conformation and thus monitor unfolding of the target protein. Free solution capillary electrophoresis achieves molecular separations without mass transfer between mobile and stationary phases and without retardation by a solid gel matrix. This separation, therefore, depend on the intrinsic properties of the sample but accurate measurement of temperature is essential. The technique allows estimation of the apparent thermodynamics parameters (Gibbs energy, enthalpy and entropy) as well as Tm(the transition midpoint of thermal unfolding) and, uniquely can show the population distribution of mobility states. It is reportedly as accurate as differential scanning calorimetry in the determination of apparent thermodynamic parameters. Temperature gradient gel electrophoresis involves the use of two electrode tanks and a horizontal plate with two water baths connected to provide a temperature gradient perpendicular to the direction of the electric field. The gel is connected to the electrode tanks via wicks. One of the water baths should have a refrigerator to maintain a suitable low temperature. Certain proteins are more stable at room temperature than in the refrigerator and are said to be cold labile. This cold denaturation has been well characterized for myoglobin and a few other proteins. It is a property of the protein itself and is distinct from freezing inactivation. 5. Kinetic stability Kinetic stability is distinct from (and need not correspond with) thermodynamic stability. It involves measuring the persistence of catalytic (or other biological) activity with time under adverse conditions of temperature, pH, solvents, salt concentration and so on (or, to put it another way, the progressive loss of function). In can be represented by the scheme kin N,,I Where N is the native, functional protein, I is an irreversible inactivated form and kin is the rate constant for the inactivation process. To conclude, there are many indices of protein stability. The most prominent of these are summarized in Table 1. Table 1 Principal indices of protein stability 8.1.2 Studies on Denaturation, Inactivation and Stabilizing Interactions 1. Introduction The crucial importance of a protein‘s tertiary structure, i.e. its molecular shape, has been remarked upon in previous chapters. Tertiary structure arises from interactions between the side chains (or R-groups) of the covalently-linked amino acids making up the polypeptide. It is the tertiary structure that orientates the critical residues and side chains into the correct geometrical relationship to permit function. This is not to state that a protein molecles is completely rigid, however. There is aboundant evidence that enzymes and proteins undergo slight but significant changes in shape on binding substrates or modulators. This part examines denaturation or unfolding of a protein and the nature of subsequent molecular changes leading to irreversible inaction. It also surveys exploirations of stabilizing interactionsin proteins and considers the contribution of the carbohydrate portions of glycoproteins to stability. 2. Denaturation studies Armed with the parameters described in previous section for measurement of protein stability, researchers have been able to study the loss of protein function or strucutre I a rational fashion. Understanding the causes of activity loss can help on the formulation of stabilizing strategies, since one will know what changes must be prevented. Many enzyme deactivations have been characterized in detail. A first-order exponential process describes many deactivations, since two-state transitions are often observed in reality. More complex phenomena do occur, however, but even some of these have been successfully modeled. Oligomeric proteins are more likely to undergo complex deactivation. Many monomeri proteins show two-state unfolding but there are exception. The single subunit protease zymogen, pepsinogen, undergoes a transition that is not two-state. One can observe denaturation of most proteins at high temperatures. This conformational stability of proteins is due to the (quite small) net difference between a very large number of weak stabilizing interactions and the nearly-equally large conformational entropy. This net free (Gibbs) energy of stabilization (typically 40 kj mol-1) is equivalent to that of a small number of interactions. Only a very few further interactions is sufficient to explain the greater stabilities of very stable or extremophilic proteins: a single interaction may contribute up to 25kj mol-1. Some proteins, however, will denature at low temperatures and this has been well described for metmyoglobin. There are examples where the unfolding temperature of a protein of interest decreases greatly in the presence of chaotropic agents. Finding of this sort show that different denaturing influences (where temperature, pHor chaotropic agents) act in an equivalent fashion by encouraging unfolding of the three-dimensional protein structure. Where inactivation takes place, some covalent change or alternation in the degree of association occurs instead of, together with, or in addition to, the unfolding phemomenon. Activity will be lost when the unfolding disrupts the integrity of the molecule‘s active or functional site(s). In summary, loss of a protein‘s biological activity can occur by either conformational or covalent processes. Air-liquid interfaces can also have important destructive effects on proteins, especially under conditions of rapid mixing or agitation, as has been demonstrated for a variety of proteins. Surfactants which preferentially absorb to the air-water interface can prevent inactivation, providing further eidence for this interfacial effect. 3. Deleterious chemical reactions in proteins Deamidation of glutamine and asparagine can occur at neutral to alkaline pH values while peptide bonds involving aspartic acid undergo cleavage under acidic conditions. Cysteine is prone to oxidation, as are tryptophan and methionine. Alkaline conditions lead to reduction of disulphide bonds and this is often folowed by beta-eliminations or thiol-disulphide exchange reactions. Where reducing sugars are present with free protein amino groups (N-termini or lysine residues), there may be destructive glycation of the amino functions by the reactive aldehyde or keto groups of the suagr (the Maillard Reaction). Elevated temperatures favor all of these reactions but it is important to note that aggregation and deleterious chemical reactions can occur at moderate temperatures also. (1) Deamidation of asparagine residues (2) Isomerization of prolines (3) Destructive oxidation events (4) Proteolytic processes 4. Probing the stabilizing interactions in proteins One can see from the paragraphs above that protein scientists have identified the events responsible for loss of protein function, but what of the molecular interactions maintaining a protein‘s folded structural integrity? Site directed mutagenesis permits the replacement of specific amino acid within a polypeptide chain. Use of these specific mutagenic techniques allows one to ascertain a particular amino acid‘s contribution to the overall structural integrity and stability of the protein within which that amino acid is located. The introduction (or elimination) of specific interactiona in a protein is now possible. Experiments of this sort have greatly increased understanding of the types of forces contributing to stability. Examples of the sorts of substitutions possible include replacement of tyrosine by phenylalanine to assess the contribution of the tyrosine –OH group, or exchange of aspartic with glutamic acid to extend or shorten the position of the carboxylic acid function by a distance of one –CH2- group. (examples are omitted). 5. Replacement of conserved residues: E. coli Thioredoxin Conservation of certain amino acid residues among similar proteins across different species is generally regarded as implying an important structural or functional role for the residues in question. In some cases, however, it has proven possible to replace conserved residues and still recover a functional protein. Example: E. coli thioredoxin Thioredoxins are redox proteins, which act as reducing agents and as protein disulfide reductases. They contain many disulfides, having about 100 amino acid residues and occur in many different types of organisms. The active sequence Trp-Cys32-Gly-Pro-Cys35 is highly conserved, along with a number of close-lying residues. Three of these amino acids were replaced by site-specific mutagenesis: Asp26, Pro34, and Pro76. These occur in most thioredoxins and lie near the active site disulfide. A mutant containing alanine at position 26(D26A) was more stable than the wide type but did have altered functional properties despite an insignificant change in redox potential. Wide type thioredoxin’s guanidine HCl midpoint for unfolding is 3.4 M at pH 7.0; that of the D26A mutant shifted upwards to 4.3M, coresponding to a 1.0 kcal mol-1 increasing stability…….. 6. Carbohydrate side chains and protein stability Glycolation is the covalent addition of carbohydrate residues to R-groups of amino acids within a polypeptide backbones. This post- translational modification occurs widely in eukaryotic systems and generally involves the side chains of Asn (N-linked) or Ser (O-linked) residues. Besides having a number of purposes in vivo, this carbohydrate labelling of a protein can confer quite a deal of additional stability. The hormone erythropoietin (EPO), which stimulates the production of red blood cell precursors and is an important therapeutic shows just how significant this extra stabilization can be. EPO is a 34-38 kDa protein containing three N-linked and O-linked carbohydrate side chains. Carbohydrate accounts for 40-50% of its molecular mass. The N-linked oligo-saccharides are essential for in vivo activity but the O-linked one is not. Removal of sialic acid from the N-linked oligosaccharides abolishes ?in vivo‘ activity even when the rest of the sugar moiety remains. 7. Is there a trade-off between stability and Activity? Studies of denaturation and of stabilizing interactions in proteins strongly suggest that proteins are optimized for function (e.g. an enzyme as catalyst for its specific reaction) rather than for stability (with the obvious exception of the proteins occurring in extremophilic organisms). There may be a trade-off between activity (which requires some degree of moleclar flexibility; cf. The induced-fit hypothesis) and folding stability (which demands a rigid polypeptide backbone). 8. Conclusion: It can be seen from discussion above, that some conclusions have emerged from studies of native and mutant proteins under extreme conditions. Inactivating covalent processes, which follow unfolding of the polypeptide, fall into a small number of defined reactions. Electrostatic interactions can be stabilizing in appropriate, rigid regions of the polypeptide. However, tight packing of the hydrophobic core so as to minimize cavities is perhaps the most important contributor to folded protein stability. 8.1.3 Enzymes in Organic Media 1. Introduction The successful use of non-membrane bound enzymes in biphasic aqueous-organic systems (and even in anhydrous organic solvents) was a novel and surprising development. Here the focus will be confined to the stability aspects of such studies. 2. Enzyme behavior in anhydrous organic solvents Some enzymes (and other proteins) have been observed to function perfectly well in non-aqueous media. Indeed, they show some remarkable properties in organic systems. A protein can retain its correct conformation on transfer from a hydrophilic aqueous to a hydrophobic organic solvent system. This is because a layer of bound or ―essential‖ water remain associated with the folded polypeptide, even in a ―completely dry‖ or lyophilized preparation. This minimal layer of water is sufficient to solvate the folded polypeptide. An unfolded polypeptide requires a greater number of water molecules for effective solvation; thus the folding equilibrium is shifted towards the folded form in low-water systems. This shift in equilibrium means that denaturation (unfolding) becomes a much less likely event in low-water hydrophobic systems. This is not the only characteristic of these media benefitting stability. A common feature of the inactivating covalent reactions is the participation of water. Water is present at the extremely high concentration of 55 M in aqueous systems. Despite the retention of ―essential‖ water surrounding a polypeptide in a hydrophobic medium, water‘s effective concentration is profoundly reduced and water- mediated deleterious covalent reactions are much less likely to occur. Thus, both the unfolding and inactivation events of the conventional N,,U,,I model can be markedly decreased in hydrophobic media. Klibanov has proposed certain rules to ensure that enzymes will remain active in organic solvents: (1) The solvent should be hydrophobic and show very little affinity for water. (2) Enzymes for use in organic solvents should be lyophilized from solutions with pH values corresponding to the enzyme‘s optimal pH. (3) The enzyme should be agitated vigorously or sonicated to ensure homogeneous dispersion in the organic solvent. Why are these rules important? (see Text for details) 3. Some case studies (Omitted) 4. Combined Strategies One can stabilize enzymes for use in the presence of organic solvents by strategies such as immobilization or protein engineering. Membrane-bound (as distinct from soluble) multi-subunit enzymes may also be stabilized as protein-lipid complexes by organic solvents. It was found that Cytochrome oxidase and H+-ATPase from inner mitochondrial membrane were stabilized by factors of 100 and 9, respectively when the water content of the toluene bulk phase was reduced from 13 microliters per milliliter to 3 microliters per milliliter. The judicious use of hydrophobic, non-polar solvents as alternatives to water has tremendous potential for the achievement of stabilized enzymes. 8.2 Manipulating Protein Stability 8.2.1 Use of Stabilizing Additives 1. Inroduction It has been known that inclusion of low molecular weight substances such as glycerol or sucrose in protein solutions can greatly stabilize the critical protein‘s biological activity. A variety of compounds (often used at concentrations of 1M or greater) can increase the stability of proteins in solution or those undergoing processes such as freeze drying. The range of such compounds is very wide. It includes substances as diverse as substrates (and specific ligands), salts, glycerol, sucrose, polyethylene glycol, chelating agent, reducing agents and proteins such as serum albumin. However, it took some time for the exact mechanism of this stabilization to be ascertained. Timasheff and colleagues have shown that these types of substances are preferentially excluded from the vicinity of the protein molecules, since their binding to the protein is thermodynamically unfavorable. The protein molecule is preferentially hydrated by the solvent water. Loss of the protein‘s compact, properly folded structure (denaturation) will increase the protein-solvent interface. This in return will tend to increase the degree of thermodynamically unfavorable interaction between the additive and the protein molecule.The result is that the protein molecule is stabilized by the additive. This ―preferential exclusion‖ means that there is less of the solute (additive) immediately surrounding the protein than there is in the bank solution; it does not necessarily mean that no solute molecules can penetrate the protein molecule‘s hydration shell. 2. Types of stabilizing molecules (1) Salts A particular salt exerts stabilizing or destabilizing effects on proteins depending on its position in the Hofmeister lyotropic series which relates to the effects of salts on protein solubility: ++++2+2+2+ (more stabilizing )(CH)N,NH,K, Na,Mg,Ca,Ba 344 ---2--- SO,Cl,Br,NO,ClO,SCN 4 34 The stabilizing ions ―salt out‖ hydrophobic residues in the protein, causing the adoption of more compact structure. This effect may be attributed to the increased ionic strength of the solution and to the increase in the number of water clusters around the protein. This helps prevent the unfolding which is the initial event in any protein deterioration process. Most stabilizing ions seems to act via a surface tension effect. Ions can also stabilize proteins by shielding surface charges and can act as osmolytes by affecting the bulk properties of water. Note that ammonia sulfate, which is widely used as a stabilizing ions from the Hofmeister list above, the NH4+ cation and the SO42+ anion. One can stabilize protein in solution while avoiding precipitation by adding ammonium sulfate to final concentration in the range 20~400mM. Besides a ammonium sulfate, salts containing citrate, sulfate, acetate, phosphate and quaternary ammonium ions are generally useful. Note, however, that the nature of the counterion will influence the overall effect of such compounds on protein stability. (2) Glycerol, sugars and polyethylene glycol Glycerol, sugars and polyethylene glycol are polyhydroxy compounds. They can form many hydrogen bonds and aid formation of a ―solvent shell‖ around the protein molecule that is distinct from the bulk aqueous phase. They can also increase the surface tension and viscosity of a solution. (3) Chelating agents Chelating agents prevents act to complex metal ions. By doing this, they prevent oxidation by active oxygen species. They can also prevent metal ion-induced aggregation. They can, however, remove a catalytically-essential metal ion from an active site, leading to loss of activity. (4) Reducing agents Reducing agents prevent the destructive oxidation of essential structural or functional features, but they do have potential drawbacks. The commonly used 2-mercaptoethanol can reduce disulfide bonds in proteins and also catalyzes a thiol-disulfide exchange which may lead to aggregation. 3. Basis of stabilization Volkin and Klibanov have classified stabilizing additives into three classes: (1) Specific: substrates and ligands, where the Native,Unfolded equilibrium shifts towards the native form; (2) Non-specific: neutral salts and polyhydroxyl compounds, which function as explained above; (3) Competitors: which out compete the enzyme for the inactivating reagents or remove the catalysts of deteriorative chemical reactions: examples included added protein, chelating and reducing agents. Schein stressed the importance of the hydration or solvent shell surrounding protein molecules in solution and has dividede solutes into osmolytic and ionic stabilizers. Osmolytes affect solvent viscosity and surface tension: thus they influence solvent ordering. They are not highly charged and do not affect enzyme activity up to 1M concentration. Osmolytes include polyols, sugars, polysaccharides, neutral polymers and amino acids. The principal method of protein stabilization by ionic compounds is by shielding of surface charges. 4. Use of Additives It is important to note that the additives discussed below are generally applicable as stabilizing agents for proteins but a given substance may not be effective for a particular protein. Both sucrose and PEG, for instance, are good stabilizers of invertase but have denaturing effects on lysozyme; the same additive has contrary effects on the two enzymes.One should note the stabilizing or destabilizing effects of the component ions of a salt when choosing additives; ref.6 includes a useful discussion of this topic. Osmolytes are a diverse group of substances comprising such compounds as polyols, mono-and polysaccharides, neutral polymers (such as PEG) and amino acids and their derivatives. One should use polyols and sugars at high final concentrations: typical figures range from 10-40%(w/v). Sugars are reckoned to be the best stabilizers but reducing sugars can react with protein amino groups, leading to inactivation. This problem can avoided by using non-reducing sugars or the corresponding sugar alcohols. Glycerols is a very widely used low molecular weight polyol. Its advantages include its ease of removal by dialysis and its noninterference with ion exchange chromatography. However, glycerol suffers from two significant disadvantages: it is a good bacterial substrate and it greatly lowers the glass transition temperature of material to be preserved by lyophilization or drying. The 5-carbon sugar alcohol, xylitol, can often replace glycerol; it can be recycled from buffers and is not a convenient food source for bacteria. Polymers such as PEG are generally added to a final concentration of 1~15%(w/v). They increase the viscosity of the single phase solvent system and thereby help prevent aggregation. Note, however, that higher polymer concentrations will promote the development of a two phase system. The protein of interest will concentrate in one of these phases and this may actually lead to aggregation. Amino acids with no net charge, notably glycine and alanine, can act as stabilizers if used in the range 20~500 mM. Amino acids and derivatives occur as osmolytes in nature. 5. Substrate and specific ligands Addition of specific substrate, cofactors or competitive (reversible) inhibitors to purified proteins can often exert great stabilizing effects. Occupation of the target protein‘s binding or active site(s) by these substances leads to minor but significant conformational changes in the polypeptide backbone. The protein adopts a more tightly folded conformation, reducing any tendency to unfold and (sometimes) rendering it less prone to proteolytic degradation. Occlusion of the protein‘s active site(s) by a bound substrate molecule or reversible inhibitor will protect those amino acid side chain which are critical for function. A starch degrading amyloglucosidase enzyme stoed in the presence of 14%(w/v) partial starch hydrolysate was 80% more stable over a 24-week period at ambient temperature than the corresponding enzyme preparation stored in the hydrolysate‘s absence. 6. Use of reducing agents and prevent of oxidation reaction The thiol group of cysteine in prone to destructive oxidative reactions. One can prevent or minimize these by using reducing agents such as 2-mercaptoethanol or DTT. One should add 2-mercaptoethanol to reach a final concentration of 5-20 mM and then keep the solution under anaerobic conditions. To achieve these anaerobic condition, one can gently bubble an inert gas such as nitrogen through the solution and fill it to the brim of a screwcap containers to minimize headspace and the chances of gaseous exchange. DTT is effective at lower concentrations: usually 0.5~1.0 mM will be suffice. DTT can act as a denaturant at higher temperature and is not very soluble in high salt. Note that these reducing agents are themselves prone to oxidation. DTT oxidizes to form an internal disulfide which is no longer effective but which will not interfere with protein molecules. 2-mercaptoethanol, on the other hand, participates in intermolecular reactions and can form disulfides with protein thiol groups. Such thiol-disulfide exchanges are highly undesirable and may actually lead to inactivation or aggregation. Much of the oxidation of thiol groups is mediated by divalent metal ions which can activate molecular oxygen. Complexation of free metal ions can prevent destructive oxidation of thiol groups. EDTA may be used to complex metal ions. Additives can greatly enhance stability without chemical or genetic manipulation of the target protein. Using empirical knowledge and the principles described, one may devise a formulation applicable to many different proteins rather than just one. This is especially useful in drug applications, where the need to satisfied regulatory requirements may work against a chemically- or genetically-modified derivative. Of course, one should note that operations such as dilution, dialysis, buffer exchange or rehydration of a dry preparations will remove the stabilizing influences. One has an achieved stability only in a particular context or ambience. In contrast, engineered proteins such as subtilisins show increased stability under many different conditions. 8.2.2 Chemical Modification of Protein in Solution 1. Introduction: Scope of chemical modification Chemical modification of protein in solution is very complicated question and would require a monograph devoted entirely to this topic. Here we attempt merely to survey the nature and types of chemical modification available for the study and derivatization of enzymes and proteins. We will also describe the functional groups of proteins that are available for reaction and presents the types of reactions they undergo. Lastly, we will summarizes a selection of chemical modifications which have stabilized a variety of enzymes and proteins. Chemical modification procedure can provide important structural and functional information concerning a protein. Soluble enzymes can be chemically modified in a variety of ways so as to alter their properties. Each of the 20 amino acids occuring in proteins has a free R-group or side chain. Many of these have reactive functional groups such as the thiol group of cysteine or the amino group of lysine residues. At least 9 amino acid side chains (Cys, Lys, Asp, and Glu, Arg, His, Trp, Tyr and Met) can react with specific reagents under mild conditions to yield chemically-modified protein derivatives, often with altered properties. The aim of the chemical modification of protein is to alter the properties of interest protein (such as enhancing the stability of protein), and minimize activity loss. Thus, careful choice of modifying reagents and conditions is required. Table 8.1 lists reactive amino acid R-groups and some of the types of compounds useful for reactions with them. Chemical modification is in many ways complementary to site-directed mutagenesis and protein engineering as a methodology for the study of protein variants: (1) relatively little structural information is required concerning the target protein; (2) the experiments are often simple to carry out and protocols may be readily implemented; (3) however, chemical modification of protein is prone to a number of pitfalls (see ―Immobilization‖). Applications of chemical modification of proteins: (1) Quantitation: two of the most reactive R-groups are the thiol and amino groups, which occur on the amino acid cysteine and lysine, respectively. These group each react with a range of agents which yield some specific colored compounds. These colored compounds can be estimated quantitatively using spectrophotometer. (2) Active-site targeting: R-group specific reagents may be used to identify residues in or near the active site. The chemically- modified residues may be conveniently identified by comparing the peptide map (or protein sequence) characteristics of the chemically-modified protein with those of the native. This approach can elucidate which active site residues are involved in binding and catylysis. Cross-linking of protein subunits or adjacent proteins cab be carried out with a wide variety of reagents (see below). 2. Chemical modification and protein stabilization Mechanisms of chemical stabilization of proteins have been categorized under four headings: (1) Cross-linking (either intra- or inter-molecular) by bifunctional reagents; (2) Strengthening of hydrophobic interactions by nonpolar reagents; (3) Introduction of new polar or charged groups, leading to additional ionic or hydrogen bonds; (4) Hydrophilization of the protein surface to reduce unfavorble surface hydrophobic contacts with water. (1) Cross-linking studies Many examples of protein stability by crosslinking have been reported using a wide range of ―bridging‖ reagents. Glutaraldehyde has been used to crosslink tetrametric lactate dehydrogenase with borohydride reduction. Sixty per cent of the initial activity was recovered and 82% of the product was in tetrameric form. (2) New Polar or Surface Charged Groups Crosslinking is not a prerequisite for protein stabilization by chemical modification. Dramatic increases in stability can result merely from the alteration of surface groups. Chemical modifications other than crosslinking can have dramatic stabilizing effects. Prominent among these is the introduction of new polar or charged groups onto a protein surface. The monofunctional reagent methyl acetimidate was used to alter 17 of the 24 available lysines of pig heart lactate dehydrogenase. This acetamidination takes place with retention of charge but the Pk values rise from 10.2 to 12.5 as the simple charged amino of the native lysine residue is replaced (Fig.6.2). The modified enzyme was more resistant to heat, alkaline, Ph and even to trypsin digestion. This last finding was ascribed to the structure of the covalently-altered lysines, which is not equivalent to either lysine or arginine, the two amino acids which are cleaved by trypsin( Fig.6.2). The increases in thermal half life ranged from 8- to 50-fold, depending on the elevated temperature used. Charged retention is not, however, always required for stabilization by monofunctionals. Alpha-chymotrypsin has been alklated(using acrolein followed by borohydride reduction, resulting in retrntion of positive charges) and acylated( with acetic or succinic anhydride, leading to the neytralization or reversal of positive charges, respectively). All of these reagents react with free amino groups of the protein. Thermal stabilities increased with the degree of substitution of the available amino groups up to a maximum of 90%. Further substitution led to a dramatic drop in thermostability. The stbilizations achieved were shown to depend on the degree of modification rather than on the type of modifier used. In other words, either alkylation or acylation reactions stabilized the enzyme provided that each substituted the free amino groups on the enzyme to an equivalent degree. This suggests that the positive charges on the surface of alpha- chymotrypsin(or, at least, some of them) are not important for retention of native conformation. Similar findings were reported for alanine aminotransferase(ALT) following reaction of its free amino groups with succinic anhydride, which reverses the positive charges of protein amino groups. This modification led to a 2-fold stabilization at 37:C(measured by comparison of first-order inactivation rate constants) and to a six-times longer shelf life at 4:C(measured by an accelerated degradation methodology).These benefits depended strongly on the use of a large excess of modifier over protein. In contrast, Cupo and colleagues observed that guanidination of chymotrypsinogen lysines by O-methyl isourea led to increased stability. The final effect of this reaction resembles the acetamidination discussed above (ref.48) but here a full guanidino group is added to the lysines to yield homoarginine. Net positive charge is retained. Subsequently, the remaining available lysines were neutralized by acetylation and the resulting derivative was less stable than the native form. A superguanidinaged chymotrypsinogen was prepared by further guanidinartion of altered protein carboxyl groups and proved to be even less stable. Taken together with the results in ref.49, these findings show just how critical the correct choice of reaction chemistries and stoichiometries can be in chemical stabilization experiments. They may also reflect the different methods used to measure stability: loss of catalytic activity and hydrogen isotope in-and out-exchanges. (3) Hydrophilization of Protein Surface This has been well illustrated by the work of Mozhaev and colleagues. Their approach was to ―hydrophilize‖ enzymes by chemical modification of non-polar surface clusters. The resulting derivative will have a greater hydrophilic surface area and be better solvated. Since its interactions with the solvent will be improved, the protein will be less likely to unfold in the face of denaturing influences. Using alpha-chymotrypsin once again as target enzyme, dramatic stability enhancements(1,000-fold at 60:C) have been achieved for surface-hydrophilized derivatives arising from reductive alkylation of up to 10 amino groups. Alkylation with glyoxylic acid, followed by - cyanoborohydride reduction, introduced a number of -NCHCOO2 groups. These are much more hydrophilic than the naturally-occurring free-NH groups on the protein surface and they probably lead to 2 decreased contact between water and nonpolar clusters. Six of the fourteen lysines lie close to hydrophobic residues on the protein surface. The 1000-fold stabilization reported is the ratio of the first-order inactivation rate constants for the native and modified forms. This was a remarkable result from such a simple chemical alteration with low molecular weight compounds. (4) PEG and carbohydrate coupling There is another form of chemical modification which can benefit stability, namely the covalent attachment of large molecular weight polyhydroxy entities such as PEG (polyethylene glycol) to proteins. Coupling of PEG to proteins has been used to alter their immunogenicity and to prolong their clearance times post-injection. PEG coupling can also improve enzymes‘ solubilities and activities in organic solvents. PEG has been used to dissolve enzymes in non-aqueous solvents to open up a whole new area of enzyme chemistry. It can also lead to increased stability. Soluble carbohydrates and polysaccharides may be coupled to proteins by the cyanogen bromide method. This activates sugar hydroxyl groups for coupling to protein amino groups. This has been suggested as a general method for enzyme stabilization: carbohydrate coupling helps protected protelytic inactivation in addition to its enhancement of protein thermostability. Trypsin and alpha- and beta-amylases have been coupled to soluble dextran. All derivatives were more stable than their untreated counterparts. For example, the 60C half-life of alpha-amylase increased from 2.5 to 63 minutes following modification. The modified trypsin was less prone to autolysis. An increased degree of hydration may be an important factor in the stabilization. (5) Other strategies 3. Conclusion A wide range of protein and enzyme properties have successfully been altered by chemical modification procedures. These include solubility, catalytic activity, substrate selectivity and stability. Chemical modification can dramatically increase protein stability, with good recoveries of activity. Active sites may be protected by specific ligands, if required, but some degree of inactivation is always a possibility. Partial loss of biological activity may be due either to decreased activity of all the enzyme/protein molecules in the system or to total inactivation of some of the molecules present. It can be difficult to distinguish between these alternatives. Lessened activity among the entire population of molecules often leads to altered kinetic parameters or pH profiles, while a decreased turnover number accompanied by an unchanged Km suggests the presence of a molecular fraction with unaltered activities. Complete loss of biological activity following a given modification reaction is usually interpreted in terms of the targetted residue(s) being essential for activity. Despite these reservations, chemical modification may be carried out and characterized quite quickly. The information gained is ueful for further rounds of chemical modification or for suggesting target sites for site-directed mutation of the protein of interest. The ability to create non-protein amino acid derivatives by chemical modification usually complements the scope of mutational/protein engineering strategies for the study or manipulation of proteins. Meanwhile, mutagenesis and expression techniques continue to improve while chemical techniques are likewise becoming more sophisticated. Very many reagents satisfy the main criteria of a useful protein modifier, namely a high specificity for one type of amino acid residue within a protein molecule and an ability to react with that residue under mild conditions of pH and solvent composition. It is likely that these powerful and complementary strategies will be increasingly combined in the future. 8.2.3 Immobilization The immobilization of enzymes and proteins onto insoluble materials forms the basis of many biotechnological processes and analytical devices. It is in its own right an extremely active field of applied study, so only the functional stability aspects of enzyme and protein immobilization will be considered here. There are four basic immobilization processes, namely: physical adsorption, covalent binding, copolymerization and microencapsulation. Each of these methods can bring about increased stability of a protein or enzyme. It is important to remember that immobilization is often performed for other reasons, such as ease of enzyme-product separation, or enzyme recyclability, and not primarily to stabilize the protein or enzyme of interest. 8.2.4 Protein Engineering See: related chapter 8.2.5 Long-term Storage of Proteins 1. Introduction The laboratory scientists and biotechnologist will often need to store an isolated or purified protein for varying lengths of time. If the protein is an object of study, it will take some time to ascertain its properties. If it is a commercial end product, or finds use as a tool in some procedure, it will likely be used in small quantities over an extended period. The protein must retain as much as possible of its original, post-purification, biological (or functional) activity throughout this time. The storage period or ―shelf life‖ can range from a few days to more than one year. The protein‘s long-term or kinetic stability becomes critically important under these conditions. Shelf life can depend on the nature of the protein and on the storage conditions. How one can prevent deterioration due to microbial contamination and proteolysis and that correct use of low temperature for extended storage are the focus of this section. It also considers drying and freezes drying as processes for long-term protein preservation. Of course, one cannot guarantee that any or all of these necessarily-broad recommendations will ―work‖ for a given protein, or that a particular stabilization factor will result from a procedure that seems to ―work‖. 2. Prevention of microbial contamination Microbial contamination can lead to significant losses of a pure protein by proteolysis. One should always aim to avoid it in the first place. (1) Addition of antimicrobial compounds such as sodium azide or thiomersal (sodium merthiolate, a mercury-containing compound) can prevent microbial growth. Add sodium azide to a final concentration of 0.1%(w/v) or thiomersal to a final concentration of 0.01%(w/v). Azide will inactivate oxygen-binding proteins such as hemoglobin or peroxidase. (2) Where one desires sterility but must avoid use of the toxic compounds above, filtration offers a useful alternative. A filter of pore size 0.22 micrometers will exclude all bacteria; indeed, this method is used in industry to sterilize labile materials which cannot be autoclaved or irradiated. 3. Avoidance of proteolysis It can be difficult to remove proteases completely during purification of a target protein. Unless the object protein is completely pure, even tiny amounts of contaminating proteolytic enzymes can cause serious losses of activity during extended storage periods. The molecular diversity of proteases complicates the situation: there are exopeptidase and endopeptidase. In addition, there are four types of proteases classified by their molecular reaction mechanisms: the serine, cysteine (or thiol), acid and metallo-proteases. Use of EDTA in the concnetration range 2-5 mM should complex the divalent metal ions essential for metalloprotease action. Pepstatin A is a potent but reversible inhibitor of acid proteases. It is used at concentrations around 0.1 micromolar, as are similar protease inhibitors. The compound phenylmethylsulphonyl fluoride (PMSF) reacts irreversibly with the essential serine in the active site of serine proteases, inactivating them. It can also act on some thiol proteases. It is typically used at a final concentration of 0.5-1.0 mM, following dissolution in a solvent such as acetone. Before addition, of course, one must ensure that none of these compounds will adversely affect the protein of interest. If the protein of interest is itself a proteolytic enzyme, use of protease inhibitors is not feasible. One may need to store such a protein in dried form or as a freeze-dried preparation. Alternatively, one can place it in a solution with a pH value far removed from the protease‘s optimum pH. Trypsin, for example, is most active at mildly alkaline pH values. Daily stock solution of trypsin are often prepared in 1mM HCl, where the very acid pH value renders the enzyme effectively incapable of catalysis. This helps prevent autolysis during the course of the experiment. The enzyme molecule does not inactivate under these conditions and is fully active on dilution into a suitable assay solution. 4. Extremely dilute solutions Very dilute protein solutions are highly prone to inactivation. This is especially true of oligomeric proteins where dissociation of subunits can occur at low concentration. The individual polypeptide chain comprising the oligomer may lack activity alone and/or may denature with consequent loss of activity. Protein solutions of concentration less -1than 1-2 mg ml should be concentrated as rapidly as possible by ultrafiltration, or by reverse osmosis using solid sucrose or polyethylene glycol. Where rapid concentration is not possible, one can prevent inactivation by addition of an exogenous protein such as bovine serum -1albumin (BSA), typically to a final concentration of 1mg ml. 5. Low temperature storage Refrigeration at 4-6:C will often suffice to preserve a protein‘s biological activity, provided microbial contamination and proteolysis are prevented. Many proteins are supplied commercially in 50% glycerol or as slurries in approximately 3 M ammonium sulphate. Freezing of such preparations is unnecessary and should be avoided. Some proteins can deteriorate at ―refrigerator‖ temperature and require storage at temperature lower than 0:C. Usually, temperatures between –18: and –20:C will allow for stable storage. Sometimes, however, it may be necessary to use temperature below –20:C. Most protein solutions will freeze solid at temperature below 0:C. The events occurring during freezing of a protein-containing mixture or biological system are much more complex than the simple macroscopic phase change suggests. Differential freezing of particular components of the mixture can lead to enormous concentration effects and to dramatic changes of pH at low temperatures. These chemical processes can lead, in turn, to a notable degree of protein inactivation. Prevention of freezing will, of course, avoid freezing damage. It is possible to undercool liquid without freezing by preventing the nucleation of ice crystals. This means that protein can be stored well below 0:C in the liquid phase. This method is very useful for small volumes of valuable proteins. It avoid the need to use additives and is more economical than freeze drying. 6. Drying for stable storage The advantages of water removal as a protein storage/stabilization strategy are many. Water participates directly in many of the deleterious chemical reactions and in proteolytic processes. In any case, it provides a medium for molecular movement and interactions. For these reasons, removal of water effectively prevents deterioration of the protein. A dried preparation will be much less bulky than the original solution and can conveniently be stored in a laboratory freezer or refrigerator (or perhaps even at room temperature). When one wishes to use the protein preparation, one can rehydrate it simply by addition of an appropriate volume of pure water or a suitable buffer solution. 7. Freezing drying Lyophilization, or freeze drying, is a method for the preservation of labile materials in a dehydrated form. It can particularly suitable for high value biomolecules such as proteins. The process involves the removal of bulk water from a frozen protein solution by sublimation under vacuum with gentle heating (primary drying). This is followed by controlled heating to more elevated temperatures for removal of the remaining ―bound water‖ from the protein preparation (secondary drying). Residual moisture levels are often lower than 1%. If the freeze drying operation is carried out correctly, the protein will preserve all or most of its initial biological activity of the protein in question. 8.3 Conclusion This chapter has tried to show that many different approaches have contributed to our understanding of, and ability to manipulate, protein stability. It is likely that such cross-fertilization will continue in the future. Advances in determining three-dimensional protein structure will aid molecular modeling and rational design approaches. Mutagenesis and expression techniques continue to improve while chemical techniques are likely becoming more sophisticated. It is likely that these powerful and complementary strategies will be increasingly combined in the future. Available stabilization strategies include immobilization, use of additive, chemical modification and protein engineering. (see Table). Towards an exciting future? Chapter 9 Functional Diversity of Proteins Keypoints: 1. How does the structure of proteins relate to the function for which they were designed? 2. How cells design protein (from the evolutionary viewpoint)? 9.1 Targeting and functional diversity The cell is a highly organized factory in which the constituent parts are assembled in different locations and specialized machinery exists for specific purposes. Thus single-cell organisms are compartmentalized so that specific reactions occur in unique locations. In multicellular organisms the localization of reactions is even greater. The workers in the biochemical factory of the organism are the proteins. 1. Proteins are directed to the regions where they are utilized Our first consideration is how proteins get to their final destination, that is, the locations where they function. All proteins are made in the cytoplasm, but their final location depends on a variety of signals. All proteins are made on ribosomes. Except for a small number of ribosomes located inside the organelles themselves, the vast majority of proteins are made on ribosome in the cytosol. Some of the ribosome are freely floating in the cytosol, and some are attached to the endoplasmic reticulum. The ribosomes that remain free account for the proteins that are targeted to locations in the cytosol, the nucleus, the peroxisomes, the mitochondria, and the chloroplasts. Ribosome that are bound to the endoplasmic reticulum make proteins that deposited in the lumen of the endoplasmic reticulum. From there the newly synthesized proteins may be transferred to the Golgi apparatus while undergoing modifications of various sorts. At some point, parts of the Golgi pinch off, and the modified proteins that do not remain in the Golgi are transferred to specific locations such as the lysosomes, the plasma membrane, and the secretory granules. Those proteins targeted to the secretory granules are eventually exported. 2. Classification of proteins according to location emphasizes functionality Because of the great structural and functional diversity of proteins, it is difficult to capture the important features or the whole range of them within any one classification scheme. Here we will classify proteins according to the location they occupy when they are fully functional. This is useful classification scheme because it emphasizes functional interrelatedness---proteins that go together work together. (Table) 3. Protein structure is suited to protein function we have seen that highly elongated fibrous proteins are well suited for compartmentalization, for giving stable form to organellar and cellular structures, and for processes involving movement of the organism. Because of their generally low mobility, fibrous proteins are rarely associated with enzyme activity or used for transport purposes. For those functions, globular proteins are more suitable. In this section, we will consider two classical examples of protein assemblages that are ideally designed for the roles they play in the cell: hemoiglobin and the skeletal muscle system. 9.2 Hemoglobin---an allosteric oxygen-binding protein Homoglobin is the best known transport protein. Its chief function is to pick up oxygen in the lungs, where it is plentiful, and deliver it to tissues throughout in body. A central feature of homoglobin is a water-free pocket for the heme, with its central iron atom located where oxygen is bound. The hydrophobic character of the heme binding cavity is dicated by the apolar side chains that line it. This is particularly suitable environment for binding the hydrophobic porphyrin ring and where iron can bind oxygen reversible without itself being oxidized to Fe3+. Hemoglobin consists of two , subunits, each with 141 amino acids, and two , subunits, each with 146 amino acids. Each subunit is capable of binding a single molecules of oxygen. In muscle cells a reserve oxygen store is provided by the myoglobin molecule, which is similar in strucutre to hemogobin but exits as a monomer. While the components of myoglobin and hemoglobin are remarkably similar, their physiological responses are very different. On a weight basis, each molecule binds about the same amount of oxygen at high oxygen tension (pressure). At low oxygen tensions, however, hemoglobin gives up its oxygen much more readily. These differences are reflected in the oxygen-binding curves of the purified proteins in aqueous solution(Fig). The oxygen-binding curve for myoglobin(Mb) is hyperbolic in shape, as would be expected for a simple one-to-one association of myoglobin and oxygen: Mb+O2 •MbO2 Kf=[MbO2]/[Mb][O2]=equilibrium formation constant If y is the fraction of myoglobin molecules saturated, and if we express the oxygen concnetration in terms of the partial pressure of oxygen [O2], then Kf=y/[1-y][O2] and y=Kf[O2]/(1+Kf[O2] This is the equation of a hyperbola. Hemoglobin(Hb) hehaves differently. Its sigmoidal binding curve can be fitted by an association-constant expression with a greater-than-first-power dependence on the oxygen concentration: Kf=[HbO2]/[Hb][O2] and y=KfO2n/(1+KfO2n) Under physiological conditions the value of n is around 2.8, indicating that the binding of oxygen molecules to the four heme in hemoglobin is not independent and binding to any one heme is affected by the state of the other three hemes. The first oxygen attaches itself with the lowest affinity, and successive oxygen are bound with a higher affinity. The exact value of n for hemoglobin is a function of the extent of oxygen binding as well as the pressure of other factors. In general, a value of n,1 indicates cooperative binding (or positive cooperativity) between small-molecule ligands, a value of n,1 indicates anticooperative binding (or negative cooperativity), and a value of n=1 indicates no cooperativity. 9.3 Muscle---an aggregate of proteins involved in contraction 9.4 Protein diversification as a result of evolutionary pressures Chapter 10 Proteins in Solution and in Membranes 10.1 Introduction 10.2 Physical and chemical properties of soluble proteins There are great differences in physical, chemical and biological roles between native folded proteins and unfolded proteins. Due to the compactness of folded conformation, the diffusion rate of native proteins is very rapid. The individual domains of proteins are relatively resistant to proteases, which is frequently used as a criterion for whether a protein is folded. Multidomain proteins often can be cleaved between domains. Some domains are cleaved by proteases at peptide bonds in mobile surface loops, but the folded structures generally remain intact. If diassociated, the fragments often recombine spontaneously under the appropriate conditions to regenerate the folded structure. The folded conformation places the atoms of a protein in unique environments that often markedly affect their physical and chemical properties. Tow or more functional groups are often held in proximate by the folded conformation, making their effective concentrations relative to each others so high that reactions occur between them that would be negligible if the functional groups were on separate molecules. Many of these properties are not evident when proteins are crystallized, but appear in solution or in membrane where the proteins are more flexible. Nevertheless, knowing the crystal structure of a protein is necessary to understand its properties under other conditions. 10.2.1 Aqueous solubility The solubility of proteins in aqueous solutions vary enormously. Some proteins are so soluble in water that they can compose up to 35% of the volume of a saturated solution. Others, especially structural proteins, are essentially insoluble under physiological conditions and exist normally as solids, aggregated into complexes of varying sizes and specificity. Many proteins that are relatively insoluble in water are sequestered into membranes. The solubility of a protein in water is determined by its free energy when surrounded by aqueous solvent relative to its free energy when interacting in an amorphous or ordered solid state with any other molecules that might be present, or when immersed in membrane. This is a very complex situation, for which no quantitative explanations of protein solubility are available. The interactions of a protein molecule with solvent or with other molecules are determined primarily by its surface. The most favorable interactions with aqueous solvent are provided by charged and polar groups of the hydrophilic side chains. The surfaces of most water-soluble globular proteins are covered uniformly be charged and polar groups, and their solubilities are governed primarily by the interactions of the polar groups with water.structural proteins also have polar surfaces, but they interact with other protein molecules more avidly than they do with water. Membrane proteins are more complex; their interactions with membranes are described later. The solubility of a globular protein in water generally increases at pH values farther away from its isoelectric point. The greater the net charge on the protein molecule, the greater the electrostatic repulsions between molecules, which tends to keep them in solution. Most proteins unfold at some pH value, however, often with drastic consequences for their solubility, because undoloding exposes many nonpolar surface areas to the solvent. Most proteins can be solubilized in aqueous solutions by adding detergents or denaturants such as urea or guanidinium salts, but the proteins are then usually unfolded. The solubilities of globular protein are affected by the addition of cosolvents, especially salts. (see discussion above). Organic solvents also tend to decrease the solubility of proteins, primarily by lowering the dielectric constant of the solvent. Polar interactions between the solvent and the protein surface are consequently less favorable. The stability of the folded state is also lowered, however, so organic solvents tend to denature proteins. Other polymers also tend to decrease the solubility of proteins. 10.2.2 Hydrodynamic properties in aqueous solution 1. Diffusion (see: other chapter) Molecules undergo random rotation and translation because of Brownian motion, which subjects them to repeated collisions with the atoms of their environment. 2. Sedimentation analysis(see: other chapter) The hydrodynamic properties of protein molecules are often measured by their sedimentation coefficient, the rate at which they sediment in a gravitational field. The rate dr/dt of sedimentation in a centrifugal field, where r is the radius at which the protein is situated and t is time, is given by 2 dr/dt=[(Mw(1-,,))/Nf],r A Mw: molecules weight; ,: its partial specific volume; ,: the density of solution; N: Avogardro‘s number; f: its translational frictional A coefficient; ,: the radial velocity of the rotor in radians per second. 3. Gel filtration 4. Rotation 10.2.3 Spectral properties The various environments of the chromophores of a folded protein and the unique stereochemistry of the polypeptide chain affect their spectral properties in various ways. These can be used to characterize and to follow changes in the folded conformation in solution. 1. Absorbance Absorbance of UV light by proteins is not very sensitive to thei conformation or environments, except for that by the aromatic rings of Phe, Tyr, and Trp residues. The spectral properties of the aromatic residue reflect their environment. Their absorbance spectra are shifted somewhat to longer wavelengths in a nonpolar environment such as the interior of a protein. The absorbance spectra of the aromatic groups consequently can be used to determine their average exposure to water. 2. Fluorescence Fluorescence by the aromatic side chains is much more sensitive to their environment than is absorbance, but it varies in an unpredictable manner. The quantum yield may be either increased or decreased by folding, so a folded protein can have either greater or less fluorescence than the unfolded form. The magnitude of the fluorescence is not very informative in itself, but it can serve as a sensitive probe of any perturbations of the folded state. Fluorescence by a protein is especially complex when there is more than one aromatic side chain. The close proximity of aromatic groups in a folded protein usually results in very efficient energy transfer between them. …… 3. Circular dichroism The CD and optical rotary dispersion(ORD) spectra of a protein are very sensitive to its conformation. In the far-UV region (below 250nm), these spectral characteristics are determined primarily by the polypeptide backbone conformation, especially its secondary structure. The spectrum of a protein of known structure is usually close to that expected from the average of the spectra of ,-helice, ,-sheets, and irregular conformations of model polypeptides, weighted by the fraction of the polypeptide chain in each conformation. Consequently, CD spectra can be used to estimate the relative proportions of the various types of secondary structure in a protein. Early methods interpreted he CD spectrum in terms of the model spectra of ,-helice, ,-sheets, and irregular conformations; more recent procedures use spectra of a number of proteins of known structure to fit the spectrum being analyzed. As long as the unknown spectrum does not have any unique features, fitting it with actual protein spectra usually gives the most meaningful interpretation. However, other chromophores, especially aromatic rings, can contribute significantly to the far-UV spectrum of a protein. Recently, Infrared and Raman spectroscopy are being developed to measure protein secondary structure in solution. 10.2.4 Ionization The folded conformation of protein have a variety of effects on the ionization of their polar groups. Many charged groups are brought into close proximity on the surface of a folded protein, so ionization of groups that would increase the net charge may be hindered. This general electrostatic effect influences the ionization of all the groups. Specific interactions, such as hydrogen bonding or salt bridging, also occur and primarily affect the ionization of particular groups. The kPa values of groups can be influenced by many environmental and electrostatic effects in small molecules. The variety of environments in folded proteins can produce very unusual ionization properties. The pKa values of residues of one type can vary widely within a single protein, Often over a range of 3-4 pH units, because of their different environments. Understanding and simulating electrostatic effects in the heterogeneous environments of a folded protein immersed in water or a membrane are much more complex than in a homogeneous liquid, where a simple dielectric constant can describe the effect of the environment. Detailed modeling of electrostatic effects in proteins requires consideration of all the atoms and charges of both the protein and the solvent, plus their atomic polarizabilities. The complexity of folded protein structures prevents such analysis, and simpler approximate models are usually used. Electrostatic effects in proteins are also complicated by the presence of centurions in the aqueous solvent and by binding of ions by the proteins. Consequently, it is impossible at present to predict accurately the ionization behavior of any one group or the titration of the total protein, but progress is being made. 10.2.5 Chemical properties The unique environments of reactive groups in folded proteins can substantially affect their chemical properties. (omitted) 10.3 Proteins in membranes Membranes provide a physical and insulating barrier between the cell interior and its environment; they also divide eukaryotic cells into compartments. (The basic structure of a membrane). The basis of membrane structure is the amphiphilic structure of the lipid molecules. Natural membranes vary in their lipid compositions, also do the two layers of their bilayer in some cases. Protein typically compose 50%of the mass of most natural membranes, but this can vary between as little as 25% and as great as 75%. The proteins mediate various functions of the membrane such as transport of appropriate molecules into or out of the cell, catalysis of chemical reactions, receiving and transducing chemical signals from the cells environment, and maintaining the membrane structure. Membrane proteins are no less important biologically than those that are water soluble, but they have not been as thoroughly studied for simple technical reasons. Membrane proteins have amphipathic structures that reflect the membrane in which they reside. They have both polar surfaces that interact with the aqueous solution and with the lipid head groups, and nonpolar surface that interact with the nonpolar interior of the lipid bilayer. Consequently, they are soluble neither in aqueous solution nor in nonpolar solvents. They can be manipulated and studied only when immersed in a lipid bilayer or a detergent micelle. 1. Association with membranes Different proteins associate with membranes to varying extents, depending on what fraction of the polypeptide chain is immersed in the membrane bilyer. (1) Integral membrane proteins (2) Nonintegral membrane proteins: water soluble, a. to be anchored to the membrane only by fatty acid chains attached covalently by their polar ends to the protein b. other proteins are associated with membrane by noncovalent interactions with the exposed surfaces of integral membrane surface. 2. Structures of integral membrane proteins Membrane proteins do not readily form three-dimensional crystals, primarily because of the membrane lipids or detergents that necessarily bound to their nonpolar surfaces. These technical problems have been solved so recently, primarily by using detergents with short chains, that the three-dimemsional structures of only three membrane proteins are known in details. Two are photosynthetic reaction centers from related bacteria, and their structures are closely similar (fig). The other is the bacterial outer membrane protein, porin, which differs markedly from the reaction center proteins. a. Photosynthetic Reaction Centers b. Porin 3. Identifying amino acid sequences likely to transverse membranes The integral membrane proteins of known structure are not markedly different in structure or amino acid composition from water-soluble proteins, except that they are slightly more hydrophobic. They differ mainly in the nature of the amino acid side chains that are on part of their surfaces. Those side chains of membrane proteins that are on the surface in contact with the membrane bilayer are less polar than the protein interior, whereas the surfaces of water-soluble proteins are much more polar than the interior. Those observations make it likely that segments of polypeptide chains that traverse membranes could be identified from their amino acid sequences alone. 4. Dynamic behavior in membranes Membrane proteins generally diffuse rapidly in the two-dimemsional -102plane of the membrane, with diffusion coefficients of about 10 cm/s, unless they are interacting with other molecules inside or outside the membrane. They usually retain their vertical orientation in the membrane, however, and do not flip between the two surfaces. The membrane lipids move even more rapidly in the membrane planes, with -82diffusion coefficients of 10 cm/s, and only very infrequently do they move from one side of the bilayer to the other. Proteins in a membrane generally induce disorder in the lipid bilayer and restrict the diffusion of neighboring lipid molecules. The restricted lipids exchange positions rapidly with others, however, indicating that the interactions between the lipids and the proteins are weak and nonspecific. Similarly, neither ordered detergent nor lipid molecules are strongly evident in the crystal structures of membrane protein. The physical state of the membrane also affects the functional properties of its proteins, but in widely varying ways. Interactions between proteins and membranes are complicated by the usual heterogeneity of the lipid in natural membranes. Proteins in membranes tend to interact with each other much more than do protein in solution. The large sizes of protein and their high concentrations in most membranes, typically at least 255 of the membrane volume, produce a large exclude volume effect. There is not much empty space in a membrane for a protein molecule to move into. Also, the orientations of the proteins are fixed relative to the membrane and to each other, fewer degrees of freedom need to be lost for them to interact specifically. Perhaps partly for these reasons, many proteins in membranes are oligomeric. 10.4 Flexibility of Protein Structure The structures of protein in crystals demonstrate varying degrees of conformational flexibility in that the electron density of any particular atom in the calculated electron density map may be spread out to varying extents. In part, this spreading reflects the existence of populations of alternative conformations. Even greater flexibility would be expected in solution, without the constraint of the crystal lattic. Indeed, it is a thermodynamic requirement that molecules the size of proteins have a substantial transient fluctuations. The most prevalent and best understood movements of atoms in molecules are the small-scale vibrations of bond lengths and angles that are detectable by infrared and Raman spectroscopy techniques. These vibrations in proteins are similar to those observed in small molecules, 1214and they occur at frequencies between 6，10/s and 10/s. On a large scale, larger movements occur, such as those of domains of large proteins that are linked together by relatively flexible ―hinge‖ segments. On the longest time scale, folded conformations are only marginally stable and therefore spontaneously undergo transient but complete -4-12unfolding with a frequency of 10-10/s, even under conditions that are optimal for stability. Protein flexibility therefore involves movements of widely varying magnitudes on a time scale that spans perhaps 26 orders of magnitude. Describing protein flexibility is not straightforward, except for that of the side chain on the protein surface, which usually can move to extents similar to those observed in small molecules and unfolded proteins. The rate at which conformational changes occur is only one aspect of protein flexibility; another is the energetics of the various conformations. There are severe constraints on the extent to which a folded protein conformation normally varies. Integral membrane proteins have varying degrees of internal flexibility, comparable to those of soluble proteins. Amino acid side chains that extend into the membranes, however, are much more restricted in their flexibility than are those of water-soluble proteins that extend into the aqueous solution. Chapter 11 Protein Engineering Protein engineering has unlimited potential to provide significant advances in science, medicine, and industry. The successful engineering of proteins requires an understanding of the basic concepts of proteins from expression to composition. The protein engineer needs to have a working knowledge of protein composition, structure, and expression. Often, the protein engineer is required to begin by finding the protein of interest hidden within a mixture of proteins. After locating the desired protein, the protein engineer must be able to clone and express it for further analysis. Before engineering the protein of interest, the characteristics of the wild-type protein must be determined from a variety of analytical methods. Mutant proteins are then produced to assess elements of the protein that are necessary for function. Within the functional sites identified, the protein engineer begins to evaluate alterations in these sites that result in the desired new properties. These new properties may include alteration in stability, catalytic activity, receptor binding, specificity, pharmacokinetic properties, or immunogenicity. The protein engineer must have a clear understanding of the required improvements and their impact on the intended use of the protein. 11.1 Introduction to Protein Engineering 1. Why engineer proteins? The term ―protein engineering‖ refers to the use of genetic manipulation techniques to alter(modify) or create proteins of interest in extremely specific ways. Substitution of a single amino acid residue at a known location can be accomplished routinely by site-specific (or site-directed) mutagenesis protocols. This approach has already had a major impact on our knowledge of protein structure and function. Site-directed mutagenesis depends on the use of synthetic oligonucleotides to direct the required mutation. It is highly specific: individual bases may be reliably altered even within a triplet codon. Non-natural amino acid substitutions have already been engineered specificially to investigated protein stability. a. The very underpinnings of the biotechnology industry with regard to proteins are founded upon protein engineering research. The applications range from creating structural motifs such as alpha- helical bundles or leucine zippers that test theories of protein folding and our current understanding of protein stability, to the production of the first- and second-generation protein products for human therapeutics or industrial products. These products include injectable proteins such as insulin, growth hormone, erythropoietin, hematopoietic cytokine granulocyte-colony stimulating factor, and viral subunits of hepatitis B, as well as enzymes for improved food production, biosensors, pollution control, and even biocomputing. The tools are now in place to obtain a remarkable understanding of protein folding and assembly and apply that knowledge to create protein-based human and animal therapeutics that are better than those currently available. The de novo design of new proteins with prescribe properties constructed from first principles will eventually result in even more useful products. b. Several fundamental questions currently being addressed by protein engineers involve catalysis, molecular recognition, protein folding, stability and protein-protein interactions. The now-classic approach of protein engineers in studying these questions to target those amino acids that potentially play functional or structural roles and then delete or replace them with alternate residues to test the role of steric constraints, hydrophobic force, electrostatics and charge, and the placement of hydrogen bonds, salt bridge, disulfide bonds, water, or metals. A database of information for a particular protein or class of proteins can be established in this fashion. In some cases, a database may give a researcher clear clues regarding how to produce a protein with a predictable change in structure or function. 2. The goals of protein engineering One of the long-term goals of these efforts is to define the fundamental rules for de novo protein design, since the ability to tailor-make a protein with a predetermined activity and structure is the ultimate dream of a protein chemist. Another more immediate goal is to sufficiently understand structure-activity relationships between a protein and a ligand to design small-molecule inhibitors of exquisite specificity. This latter goal constitutes the theme of structure-based drug design. Finally, generation of the redesigned enzymes provides insight into basic principles governing protein structure-function relationships and produces scientific reagents of practical importance. It should be clear from this description that protein engineering is both an area requiring basic research input and an empirical technology capable of being applied to broad areas of research as well as the generation of products. Not just enzymes but protein-base reagents, in general, will undergo a similar increase in demand. Some novel and cost-effective protein drugs such as erythropoietin, interferons, and human growth hormones have been brought to market, while others are in the developmental pipeline. Development of second-generation derivatives of these drugs and the discovery of new first-generation products will depend heavily upon the field of protein engineering. Other area where protein engineering principles will have significant impact are as follows: a. Macromolecular recognition: specifically how proteins recognize one another and recognize their substrates. b. Target drug delivery: specially the generation of high concentrations of a particular drug at a specified site. By designing an enzyme to convert a precursor to a form a drug to its active form, the drug will be delivered site specially. c. Bioremediation: d. Engineered biological catalysis. These are a few of the relatively short-term projects that the protein engineers are currently focusing their efforts on. The long-term goal is to provide a theoretical framework for addressing the relationship between the three-dimensional structure and the function of a protein. 11.2 Production and Analytical Characterization of Proteins 11.2.1 DNA level processes 1. Methods and rationale The current methods of recombinant DNA technology permit a protein engineer to address structure-activity relationships of proteins and enzymes in ways never before imagined. Genes or cDNAs encoding proteins of interest can be cloned from natural sources or synthesized de novo from the known protein sequence. The DNA sequences can then be genetically engineered to encode redesigned proteins with insertions, deletions, or precisely placed amino acid substitutions. The altered genes can then be introduced into appropriate heterologous expression systems to overproduce the introduced protein to reagent-level quantities and qualities. The proteins can be purified and analyzed both biochemically and biophysically to determine the effect of the function of the protein, the synthetic technology of recombinant DNA must be wed with the analytical techniques of enzymology, structural analysis, and molecular dynamics. The basic methods involved in protein engineering will now be described. a. Finding the protein of interest If the wild-type protein is not available, but its sequence has been determined, cDNA can be synthesized. Cloned into the appropriate plasmid, and expressed in the desired host. In the case of discovery research, the protein of interest may not be well-defined. If a homologous sequence (e.g., same family or different species) is known, cDNA probes from these molecules are often used to locate the oligonucleotide sequence encoding the desired protein within a cell that is producing the protein. For discovery research, homologous proteins may not be known, but a ligand or cofactor may be available. The ligand is then used to locate the protein of interest through radiolabeling and immunoprecipitation methods. Once sufficient quantities (~100ug to 1 mg) of the wide-type protein are available, it can be used to generate monoclonal antibodies, utilized for subsequent assays and purification. The antibodies produced can then be used to identify the protein of interest and its mutant. However, for research purposes, polyclonal antibodies derived from animal antisera may be sufficient to purify the desired quantities of the protein. The purified protein can be sequenced by proteolytic digestion and subsequent amino acid sequence analysis of the peptide fragments. After obtaining a partial amino acid sequence, cDNA can be generated for probing cells for expression of the protein as well as construction of plasmids for expression of the protein in foreign hosts. b. Developing recombinant DNA libraries Armed with primary sequence information and/or antibodies to a protein of interest, isolation of the natural gene and/or cDNA encoding the protein is a relatively straightforward task. (see also other related chapter) c. Mutagenesis principle Most protein engineering involves recombinant DNA methodologies, but other methods such as random mutation via DNA damaging agents or environmental pressures have been used. A gene encoding the target protein is cloned from the original source or synthesized and subcloned based on the protein sequence of interest. Once the gene for the target protein is cloned, one or many amino acids can be substituted, deleted, or inserted into the gene. Both natural and unnatural amino acids can be introduced into the gene at this point. The ready availablity of synthetic DNA provides a protein engineer with new vistas in the manipulation and selective alteration of cloned DNA. In particular, it is now feasible to change any cloned nucleotide sequence to any other desired sequences and to determine the effect of the change. One of the most powerful techniques for accomplishing such nucleotide substitutions, insertions, or deletions is through the use of synthetic DNA oligonucleotides. 2. Oligosynthesis a. The synthesis of DNA is based on the Merrifield principle of solid-phase chemistry that has previously developed for the synthesis of peptides. Activated nucleotided are sequentially added to the 5‘ hydroxyl of an oligo chain bound to an insoluble solid support by a 3‘ hydroxyl linkage. b. Automated DNA synthesizer. 3. Use of oligo as hybridization probes The availability of synthetic DNA of predetermined sequence has greatly facilitated studies of small fragments, permitting critical evaluation of the various parameters affecting the stability of short DNA/RNA duplexes.(Fig 1.3). 4. Use of oligos for site-specific mutagenesis Once a gene or cDNA has been cloned and its sequence determined, a critical analysis of its genetic structure and function frequently requires its expression in an appropriate system. Development of optimal expression systems is currently an area of intense investigation, and approaches include both cytoplasmic and secreted expression in bacteria, yeast and cultured insect and mammalian cells as well as expression in live animals. Once an assay for monitoring the expressed foreign gene product in the heterologous environment has been developed, it is then possible to probe structure-function relationship by site-specific mutagenesis. The effect o nucleotide or amino acid change has on an activity of the gene product can be determined by site-specifically modifying the cloned DNA, expressing, and then analyzing the activity of the mutated product. Efficient application of this so-called ―reverse genetics‖ in the analysis of a biological macromolecule requires prior knowledge of the relative importance of a given short sequence with respect to the rest of the genetic unit. In case where accurate crystallographic determinations have been made, the three-dimensional structure of a protein or nucleic acid may suggest functional roles for individual amino acid or nucleotide residue. Computer-graphics-assisted modeling building studies can then suggest residue replacements that could modify the activity of the gene product. This strategy is a classic one for a protein engineer. (Figure 1.4) Applications of oligo-directed mutagenesis 11.2.2 Protein Characterization 1. Methods to determine and assess the protein structure and composition a. Designing and modeling protein structure While the qualitative nature of the forces (that operate within proteins and between the protein and its substrate) is reasonable well understood, their quantitative contributions to protein function are not yet definable in most cases. However, there has been significant success in the use of computer software in the analysis of known protein structures in designing o proteins with novel functional properties. Such semiquantitative analyses often take into account functional and structural data of first- and higher-generation mutants, in addition to established structural biological and chemical theories. Protein engineering efforts to test or later enzyme function is an experimental science. Computer-based design of novel mutants makes extensive use of existing general-purpose modeling programs, INSIGHT and MIDAS. These programs allow rapid inspection and manipulation of structure available through the protein data bank. b. Expression A basic requirement for developing a genetic approach to studying protein is an efficient expression system for the protein of interest. High-level expression of modified proteins has proven to be difficult and unpredictable. The choice of an expression system will largely depend on the desired use of the expressed protein. ….. it is essential that the method of biosynthetically producing the target protein provides authentic material in reagent levels and quality. The most common step that permits the development of a successful protein engineering project is one involving the high-level production of functionally active protein. 2. Assessment of mutant proteins It is generally the goal in protein engineering projects to evaluate the effects of the specific modification(s) on the structure or function of the protein of interest. There will therefore be specific characteristics or functions which are the prime targets of the protein engineer, who will examine them in general detail. However, it is important to establish that the desired modifications, and no others, have indeed been introduced into the engineered protein. Only in this way is it possible to link the observed changes in structure or function to the newly introduced changes in the molecule. For many protein engineering projects, the wild-type protein is freely available and its structural characteristics are well understood. The DNA sequence of the wild-type and mutant protein are known, and their primary structures (amino acid sequences) are known. Clearly, therefore, the most important property of the new protein species that needs to be confirmed is its amino acid sequence. One of the most powerful indirect methods for assessing the correct incorporation of the structural alteration expected from the engineering is mass spectrometry, specially using electrospray methodology. …… Peptide mapping is a common, powerful tool used in the analysis of a protein. Cleavage of the protein by a specific enzyme leads to a series of peptides which are separated (typically by reversed phase HPLC) into a unique pattern. Each peak in such a separation is characterized and identified as a particular part of the protein sequence. Thus, if a well-characterized peptide map is available for the parent sequence, it can be predicated which features of the map should be altered in the new protein and only a few peptides need to be characterized to confirm that the expected changes in sequence are indeed present. Even though indirect evidence (that the desired alteration is present) may be abundant, the amino acid sequence of the altered peptide should be directly demonstrated to be consistent with that predicted. As noted above, the strongest link between the introduced change and the observed alterations in structure or function comes from the demonstration that no other changes have been introduced, at least in the amino acid sequence. The identity of the peptide map in all regions other than those affected by the desired change can usually be taken as good evidence that there have been no other changes. Table 1.4 shows some of the methods that might be used to investigate such changes or to confirm that no observable differences exist between the parent and wild-type protein. 11.2.3 Summary of Issues to Consider before Engineering a Protein 1. What is needed? Prior to altering the composition of the wild-type protein, the protein engineer must have a well-defined rational for mutagenesis studies. In particular, these studies fall into two primary, although not mutually exclusive, areas: structure-function analysis and reconfiguraton of the protein to provide useful properties (e.g. increased stability, longer half life, etc.). Structure-function analysis is often performed to understand the critical elements of the protein. Many of these studies involve large screens of mutants, and these mutants are often dramatically different from the wild-type protein. Another common alteration in the wild-type protein is cassette substitution or removal of several adjacent amino acids, with each mutant containing a different set of substitutions. This type of mutagenesis scan often pinpoints the critical functional domain of the protein. Of course, each of these mutants may significantly alter the protein‘s conformation, and, therefore, it is critical to determine the effects of the mutations by analytical techniques. Assuming that the protein conformation (secondary and tertiary structure) remains significantly unaltered, the observed differences in function between the mutant and wild-type molecules provide essential insight into the importance of the mutated regions in the protein‘s overall function. While these structure-function studies are often necessary before redesigning the protein, many alterations may be made a priori, especially if the wild-type protein is a member of a large well-defined family of proteins, some of which may have already been studied for structure and function. The next issue then becomes the choice of desired attributes to confer onto the protein. The most common alterations include mutations that provide greater specificity, higher binding affinity, faster rates of reaction (enzyme catalysis or binding rates), and altered clearance rates. Before proceeding with protein engineering, the behavior of the wild-type protein both in vivo and in vitro must be well understood. …… overall, the desired protein properties and potential mutagenesis sites must be determined prior to the design of a superior mutant protein. 2. Industrial issues Protein engineering holds great promise for elucidating the underlying mechanisms of protein structure, function and folding. Beyond enhancing the general understanding of proteins, proteins, protein engineering has the potential to great improve their use in industrial applications. For enzyme design, protein engineering provide the opportunity to design more efficient and stable enzymes. These enzymes can be used to catalyse complex reactions that, by standard chemical methods, are inefficient or lead to racemic mixtures resulting in impure products or intermediate. In addition, enzymes for many industrial chemical reaction should be stabilized against environmental stresses such as heat or organic solvents. In the case of therapeutic proteins, mutations in a naturally occurring protein may provide significant benefits. For example, (omitted). Finally, protein engineering provides a basis for rational drug design. By developing an understanding of protein structure and function relationships, the molecular epitopes that determine a protein‘s function can be used to develop small molecular drugs. By understanding biological responses such as signal transduction through receptor dimerization and phosphorylation, it may ultimately be possible to rationally design small-molecule agonists to replace therapeutic proteins. Achievement of this last goal will surely take protein engineering into the next century and beyond. 11.3 Protein Engineering for Stability 11.4 Engineering therapeutic antibody 11.5 Site-directed drug design Chapter 12 Proteomics 12.1 Introduction to the Proteomics 12.1.1 Proteome: a new word, a new field biology 12.1.2 The Proteome and Technology Thinking in two dimenesions Further dimensions in protein analysis Information and the proteome 12.2 Two-Dimensional Electrophoresis: The State of the Art and Future Direction 12.3 Protein Identification in Proteome Projects 12.4 The Importance of Protein Co- and Post- Translational Modifications in Proteome Projects 12.5 Proteome Databases 12.6 Interfacing and Integrating Databases 12.7 Large-scale Comparative Protein Modelling 12.8 Applications of Proteomics 蛋白质组研究的兴起 在后基因组时代～研究的重点已从揭示遗传信息转移到功能基因组学上来。但是～由于生物功能主要体现者是蛋白质～而蛋白质有其自身特有的活动规律。如蛋白质修饰加工、转运定位、结构变化、蛋白质与蛋白质间、蛋白质与其他生物大分子的相互作用等～均无法在基因组水平上获得。因为基因组学有样的局限性～促使人们从整体水平上探讨细胞蛋白质的组成及其活动规律。 蛋白质组和蛋白质组学概念的提出 1994年～澳大利亚Macquarie大学的Wilkins和Williams首先提出了蛋白质组, Proteome,的概念～早期定义为:微生物基因组 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 达的整套蛋白质～在多细胞微生物中～整套蛋白质指一种组织或细胞表达的蛋白质～后来定义为:一个基因组所表达的蛋白质。但是～从基因表达的角度来看～蛋白质组的蛋白质数目总是少于基因组的基因数目。从蛋白质修饰的角度来看～蛋白质组的蛋白质数却多于其相应的ORF数目～因为mRNA的剪切和编辑可使一个ORF产生数种蛋白质～蛋白质翻译后的修饰～如糖基化、磷酸化同样增加蛋白质的种类～氨基酸序列一致的一级结构在一定条件下可以形成功能完全不一样的具有不同空间结构的蛋白质～如朊病毒。故"蛋白质组内蛋白质数目要多于基因组内的基因数目"。现在蛋白质组的概念为:在一种细胞内存在的全部蛋白质。但是由于蛋白质组在不同的时间、不同的条件具有不同的蛋白质组分～而且衡量是否是蛋白质组的全部蛋白质尚缺乏必要的尺度。所以～欲得到"细胞内存在的所有蛋白质"是不可能的。 蛋白质组学,Proteomics,是以蛋白质组为研究对象的新的研究领域。它可分为:?表达蛋白质组学,expression proteomic即把细胞、组织中的蛋白～建立蛋白定量表达图谱～或扫描EST图。该方法依赖2,D凝胶图和图像分析技术～而且在整个蛋白质组水平上提供了研究细胞通路～以及疾病、药物相互作用和一些生物刺激引起的功能紊乱的可能性。 ?细胞图谱蛋白质组学,cell,map proteomics,:即确定蛋白质在亚细胞结构中的位臵,通过纯化细胞器或用质谱仪鉴定蛋白复合物组成等～来确定蛋白质-蛋白质的相互作用。Humphery,Smith等总结了基因组结果后提出了"功能蛋白质组, Functional Proteome,"的新概念。即细胞内与某个功能有关或在某种条件下的一群蛋白质。鉴于此～我国学者李伯良提出了"功能蛋白质组学,Functional Proteomics,"的概念。即把"功能蛋白质组"作为主要研究内容。这一概念的提出～为蛋白质组研究的可能性奠定了理论基础。 蛋白质组研究的理论基础 蛋白质组分析主要基于3条理由: ? 从mRNA表达水平并不能预测蛋白表达水平。有人研究了mRNA和蛋白质表达的关系～以处于对数生长期的啤酒酵母为研究对象～mRNA的表达由 SAGE,serial analysis of gene expression,频率表指示～同位素标记酵母蛋白～共选择80个基因～结果没有发现翻译和转录丰度有明显相关。 ? 蛋白质的动态修饰和加工并非必须来自基因序列。在mRNA水平上有许多细胞调节过程是难以观察到的～因为许多调节是在蛋白质的结构域中发生的。许多蛋白只有与其他分子结合后才有功能～蛋白的这种修饰是动态的、可逆的～这种蛋白修饰的种类和部位通常不能由基因序列决定 ? 蛋白质组是动态反映生物系统所处的状态。细胞周期的特定时期、分化的不同阶段、对应的生长和营养状况、温度、应激和病理状态～这些状态所对应的蛋白质组是有差异的。蛋白质组学的研究可望提供精确、详细的有关细胞或组织状况的分子描述。因为诸如蛋白质合成、降解、加工、修饰的调控过程只有通过蛋白质的直接分析才能揭示。 蛋白质组学用于医疗研究的重点 蛋白质组方面的研究～将帮助人们寻找到一些用于医疗的可识别蛋白～这些蛋白可作为诊断标记或作为诊断靶分子提供给从事医药和诊断研究的机构。研究主要有以下五个方面: 1(癌症针对研究的肿瘤类型包括:食道、肺、结肠、前列腺、胰腺、乳房以及成神经细胞 瘤。 2(神经性疾病研究方向主要包括:脑损伤和感染性蛋白质疾病～如克雅氏病,CJD,、牛海绵状脑病,BSE,、帕金森氏病。 3(器官移植排异蛋白质组研究将寻求一种体外检测的方法～用于人体器官,心脏、肝、肺或肾,移植后的过敏和慢性排异性反应。 4(心血管疾病列入研究的心血管疾病有心力衰竭、高血压合肥大型心肌炎。 5(糖尿病、肥胖症通过蛋白质组学方法对于肥胖症及糖尿病相关的多肽进行识别～作为潜在的识别分子和治疗靶象。 Sites for Proteome: ExPASy Home page Site Map Search ExPASy Contact us Hosted by PKU China Mirror sites: Australia Canada Korea Switzerland The Taiwanese ExPASy site, tw.expasy.org, is unavailable for maintenance. Please bookmark the addresses of the other mirror sites and use one of them during the downtime. We apologize for any inconvenience caused. SWISS-2DPAGE Two-dimensional polyacrylamide gel electrophoresis database SWISS-2DPAGE contains data on proteins identified on various 2-D PAGE reference maps. You can locate these proteins on the 2-D PAGE maps or display the region of a 2-D PAGE map where one might expect to find a protein from SWISS-PROT [More details / References / Disclaimer]. Release 13.0, December 2000 and updates up to 27-Feb-2001 (contains 772 entries in 31 reference maps from human, mouse, Arabidopsis thaliana, Dictyostelium discoideum, Escherichia coli and Saccharomyces cerevisiae). [Search][Documents][Services][Software][Related servers][Other databases][Job openings] , by description (DE lines) or by ID , User manual , by accession number (AC lines) , Release notes (December 19, 2000) , by clicking on a spot: select one of our 2-D , Protocols: PAGE reference maps, click on a spot and o Technical information about 2-D then get the corresponding information from PAGE (IPG's, silver staining, the SWISS-2DPAGE database. protocols, etc) , by author (RA lines) o High performance 2-D gel , by spot serial number (2D lines) comparison , by full text search , 2-D PAGE maps published: , SRS, searching in SWISS-2DPAGE using the o Human CSF, ELC, HEPG2, Sequence Retrieval System HEPG2SP, LIVER, LYMPHOMA, , retrieve in a table all the protein entries PLASMA, PLATELET, RBC, U937, identified on a given reference map CEC, KIDNEY. , compute estimated location on reference o Dictyostelium discoideum, maps for a user-entered sequence Escherichia coli, Saccharomyces cerevisiae. , Downloading SWISS-2DPAGE by FTP , Melanie 3 - Software package for 2-D PAGE analysis , SWISS-2DSERVICE - Get your 2-D Gels performed according to Swiss standards , Make2ddb package - A package preparing the data and the programs necessary to build a , 2-D PAGE training - attend a one week federated 2-DE database on one's own web course in Geneva site. , 2-D PAGE museum - gels run by trainees during the 2-D PAGE courses , 2D Hunt - 2-D electrophoresis web site finder , WORLD-2DPAGE - Index to other Federated 2-D PAGE databases , SWISS-PROT , SWISS-3DIMAGE , CD40Lbase , PROSITE , SWISS-MODEL Repository , Proteomics tools , ENZYME , SeqAnalRef Last modified 9/Apr/2001 by CHH ExPASy Home page Site Map Search ExPASy Contact us Hosted by PKU China Mirror sites: Australia Canada Korea Switzerland The Taiwanese ExPASy site, tw.expasy.org, is unavailable for maintenance. Please bookmark the addresses of the other mirror sites and use one of them during the downtime. We apologize for any inconvenience caused. Site Map Search ExPASy Contact us Hosted by PKU China Mirror sites: Australia Canada Korea Switzerland The Taiwanese ExPASy site, tw.expasy.org, is unavailable for maintenance. Please bookmark the addresses of the other mirror sites and use one of them during the downtime. We apologize for any inconvenience caused. ExPASy Molecular Biology Server This is the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB). This server is dedicated to the analysis of protein sequences and structures as well as 2-D PAGE (Disclaimer). 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Please bookmark the addresses of the other mirror sites and use one of them during the downtime. We apologize for any inconvenience caused. 基因组研究自从开展以来已经取得了举世瞩目的成就。 在过去几年中, 已经陆续完成了包括大肠杆菌、酿酒酵母、拟南芥,T. Arabidopsis,等十多种结构比较简单的生物的基因组DNA的全序列分析。 线虫(C.elegans)的基因组DNA测序工作已基本完成。 规模更为庞大的人类基因组计划预期在本世纪初(2003，2005年)也将完成全部基因组DNA的序列分析。 这些进展是非常令人振奋的。 但是也随之产生了新问 题 快递公司问题件快递公司问题件货款处理关于圆的周长面积重点题型关于解方程组的题及答案关于南海问题 。 大量涌出的新基因数据迫使我们不得不考虑这些基因编码的蛋白质有什么功能这个问题。 不仅如此, 蛋白质作为生物功能的主要载体～拥有自身特有的活动规律～在细胞合成蛋白质之后, 这些蛋白质往往还要经历翻译后的加工修饰、转运定位、结构变化、蛋白质与蛋白质间、蛋白质与其他生物大分子的相互作用等～ 也就是说, 一个基因对应的不是一种蛋白质而可能是几种甚至是数十种。 包容了数千甚至数万种蛋白质的细胞是如何运转的,或者说这些蛋白质在细胞内是怎样工作、如何相互作用、相互协调的,这些问题远不是基因组研究所能回答得了的。因为基因组学有这样的局限性～促使人们从整体水平上探讨细胞蛋白质的组成及其活动规律。 为了充分了解和全面认识生命活动的奥秘～90年代中期～在人类基因组研究计划的基础上～萌发了一门新兴的学科,蛋白质组学(proteomics)～即从蛋白质组的水平进一步认识生命活动的机理和疾病发生的分子机制。科学家们预测～随着人类基因组全部测序工作的完成～21世纪生命科学的研究重心将从基因组学转移到蛋白质且学～生命科学领域内一个崭新的时代,蛋白质组时代即将开始。 1994年～澳大利亚Macquarie大学的Wilkins和Williams首先提出了蛋白质组, Proteome,的概念～最早见诸于1995年7月的―Electrophoresis‖杂志上, 它是指一个有机体的全部蛋白质组成及其活动方式。早期定义为:微生物基因组表达的整套蛋白质～在多细胞微生物中～整套蛋白质指一种组织或细胞表达的蛋白质～后来定义为:一个基因组所表达的蛋白质。但是～从基因表达的角度来看～蛋白质组的蛋白质数目总是少于基因组的基因数目。从蛋白质修饰的角度来看～蛋白质组的蛋白质数却多于其相应的ORF数目～因为mRNA的剪切和编辑可使一个ORF产生数种蛋白质～蛋白质翻译后的修饰～如糖基化、磷酸化同样增加蛋白质的种类～氨基酸序列一致的一级结构在一定条件下可以形成功能完全不一样的具有不同空间结构的蛋白质～如朊病毒。故"蛋白质组内蛋白质数目要多于基因组内的基因数目"。 蛋白质组研究虽然尚处于初始阶段, 但已经取得了一些重要进展。 当前蛋白质组学的主要内容是, 在建立和发展蛋白质组研究的技术方法的同时, 进行蛋白质组分析。 对蛋白质组的分析工作大致有两个方面。 一方面, 通过二维凝胶电泳得到正常生理条件下的机体、组织或细胞的全部蛋白质的图谱, 相关数据将作为待检测机体、组织或细胞的二维参考图谱和数据库。 一系列这样的二维参考图谱和数据库已经建立并且可通过联网检索。 二维参考图谱建立的意义在于为进一步的分析工作提供基础。 蛋白质组分析的另一方面, 是比较分析在变化了的生理条件下蛋白质组所发生的变化。 如蛋白质表达量的变化、翻译后修饰的变化, 或者可能的条件下分析蛋白质在亚细胞水平上的定位的改变等。 蛋白质组学,Proteomics,是以蛋白质组为研究对象的新的研究领域。它可分为:?表达蛋白质组学,expression proteomics: 即把细胞、组织中的蛋白～建立蛋白定量表达图谱～或扫描EST图。该方法依赖2,D凝胶图和图像分析技术～而且在整个蛋白质组水平上提供了研究细胞通路～以及疾病、药物相互作用和一些生物刺激引起的功能紊乱的可能性。 ?细胞图谱蛋白质组学,cell,map proteomics,:即确定蛋白质在亚细胞结构中的位臵,通过纯化细胞器或用质谱仪鉴定蛋白复合物组成等～来确定蛋白质-蛋白质的相互作用。Humphery,Smith等总结了基因组结果后提出了"功能蛋白质组,Functional Proteome,"的新概念。即细胞内与某个功能有关或在某种条件下的一群蛋白质。鉴于此～我国学者李伯良提出了"功能蛋白质组学,Functional Proteomics,"的概念。即把"功能蛋白质组"作为主要研究内容。这一概念的提出～为蛋白质组研究的可能性奠定了理论基础。 蛋白质组分析主要基于3条理由:? 从mRNA表达水平并不能预测蛋白表达水平,? 蛋白质的动态修饰和加工并非必须来自基因序列。在mRNA水平上有许多细胞调节过程是难以观察到的～因为许多调节是在蛋白质的结构域中发生的。许多蛋白只有与其他分子结合后才有功能～蛋白的这种修饰是动态的、可逆的～这种蛋白修饰的种类和部位通常不能由基因序列决定,? 蛋白质组是动态反映生物系统所处的状态。细胞周期的特定时期、分化的不同阶段、对应的生长和营养状况、温度、应激和病理状态～这些状态所对应的蛋白质组是有差异的。蛋白质组学的研究可望提供精确、详细的有关细胞或组织状况的分子描述。因为诸如蛋白质合成、降解、加工、修饰的调控过程只有通过蛋白质的直接分析才能揭示。 蛋白质组学强调的是针对蛋白质的一个整体思路。从整体的角度看～蛋白质组研究大致可以分为两种类型:一种是针对细胞或组织的全部蛋白质～即着眼点就是整个蛋白质组,而另一种则是以一个特定的生物学问题或机制相关的全部蛋白质为着眼点～在这里整体是局部性的。针对细胞蛋白质组的完整分析的工作已经比较全面地展开～不仅如大肠杆菌、酵母等低等模式生物的蛋白质组数据库在建立之中～高等生物如水稻和小鼠等的蛋白质组研究也已开展～人类一些正常和病变细胞的蛋白质组数据库也在建立之中。与此同时～更多的蛋白质组研究工作则是将着眼点放在蛋白质组的变化或差异上～也就是通过对蛋白质组的比较分析～首先发现并去鉴定在不同生理条件下或不同外界环境条件下蛋白质组中有差异的蛋白质组分。 1999年11月在《Nature》杂志上发表了一篇用蛋白质组学方法研究蛋白质折叠的研究 论文 政研论文下载论文大学下载论文大学下载关于长拳的论文浙大论文封面下载 ～揭示了蛋白质与分子伴侣GroEL相互作用的关键结构特征。这项工作很好地体现了蛋白质组学的思想方法和技术手段的应用。Rout等 (J Cell Biol, 2000, 148:635-651)通过使用蛋白质组学的手段鉴定了完整的酵母核孔复合体所有能检测到的多肽～并系统地对每种可能的蛋白质组分在复合体内定位并定量～从而揭示了酵母核孔复合体的完整分子结构～并在此基础上揭示了其工作原理。这个工作可以说是蛋白质组学解决结构生物学问题的一个典范～为揭示其它巨大分子机器的“构造”和工作原理指出了一条新路。从近期国际上蛋白质组学研究的发展动向可以看出～揭示蛋白质之间的相互作用关系～建立相互作用关系的网络图～已成为揭示蛋白质组复杂体系与蛋白质功能模式的先导～业已成为蛋白质组学领域的研究热点。2000年初～《Science》刊载了一篇应用蛋白质组学的大规模双杂交技术研究线虫生殖器官发育的文章～初步建立了与线虫生殖发育相关的蛋白质相互作用图谱～从而为深 入研究和揭示线虫发育的机理等提供了丰富的线索。这一工作为以前专注于信号转导过程中单个蛋白质作用的科学家们提供了一个新的思路～即将整个途径的相关蛋白质一起考虑。 如果说蛋白质学刚诞生时没有得到国际生物学主流的重视～那么近两年情况已有了巨大的改变。美国国立卫生研究院,NIH,所属的国立肿瘤研究所,NCI,投入了大量经费支持蛋白质组研究。同时～NCI和美国食品与药物管理局,FDA,联合开发可用于临床的蛋白质组技术。美国能源部不久前也启动了一个蛋白质组项目～旨在研究涉及环境和能源的微生物和低等生物的蛋白质组。欧共体目前正在资助酵母蛋白质组研究。英国生物技术和生物科学研究委员会最近也资助了三个研究中心～对一些已完成或即将完成全基因组测序的 生物开展蛋白质组研究。在法国～五个研究不同模式生物的实验室得到为期三年的资助～每年约为500万美圆平均分配到基因组、转录组和蛋白质组研究中。德国也没有忽略蛋白质组研究～去年联邦政府投资了730万美圆开展蛋白质组和相关技术研究～并建立了一个蛋白质组中心。1998年澳大利亚政府着手建立第一个蛋白质组研究网APAF,Australian Proteome Analysis Facility,。APAF将为该国的有关实验室提供一流的仪器设备～并把他们整合在一起进行大规模的蛋白质组研究。我国关于蛋白质族研究的国家自然科学基金重大项目也从1999年开始启动。 蛋白质组研究领域的另一个特色是～许多实验室、公司和药厂等很早就已经开始进行与应用前景有关的蛋白质组研究。如膀胱癌、早老年痴呆症的蛋白质组研究,利用蛋白质组技术筛选疫苗等。据报道～ Myriad公司将与美国Oracle公司～日本日立股份和瑞士Friedli基金组织合作推出"蛋白质组"研究计划。由Myriad公司控股～四家公司共同投资一亿八千五百万美元成立的Myriad蛋白质组学股份有限公司将把鉴定人体中存在的30万种以上的蛋白质为目标～并力争弄清各种蛋白质之间相互作用的机制。对此～Myriad公司的首席执行官Peter Meldrum说:"我们将力争在分子水平上去揭示生命过程的奥秘"。 Myriad公司的计划分为两个部分:第一部分～是在酵母中表达人体的每一种蛋白质的同时研究这些蛋白质的相互作用,第二部分则把目标放在分析人体蛋白质复合体的组成及其中各蛋白质组分的功能及调控机制上。总之～两个方面的研究将帮助科学家们了解蛋白质如何实现正常的细胞功能以及如何抵抗疾病的侵袭。同时～Myriad公司所面临的竞争也非常激烈～曾在人类基因组计划中发挥了重要作用的Celera公司也不甘示弱～他们也早就瞄准了蛋白质组学这一非常具有吸引力的研究领域。 虽然蛋白质组学还处于一个初期发展阶段～但相信随着其不断地深入发展～蛋白质组,学,研究在揭示诸如生长、发育和代谢调控等生命活动的规律上将会有所突破～对探讨重大疾病的机理、疾病诊断、疾病防治、新药开发、植物生长发育调控机理等方面提供重要的理论基础。 本学术方向的研究内容分两个方面～第一方面是蛋白质组,学,用于医疗研究。这方面的研究～将帮助人们寻找到一些用于医疗的可识别蛋白～这些蛋白可作为诊断标记或作为诊断靶分子提供给从事医药和诊断研究的机构。研究的重点主要有以下五个方面: ,1,癌症针对研究的肿瘤类型包括:食道、肺、结肠、前列腺、胰腺、乳房以及成神经细胞 瘤。 ,2,神经性疾病研究方向主要包括:脑损伤和感染性蛋白质疾病～如克雅氏病,CJD,、牛海绵状脑病,BSE,、帕金森氏病。 ,3,器官移植排异蛋白质组研究将寻求一种体外检测的方法～用于人体器官,心脏、肝、肺或肾,移植后的过敏和慢性排异性反应。 ,4,心血管疾病列入研究的心血管疾病有心力衰竭、高血压合肥大型心肌炎。 ,5,糖尿病、肥胖症通过蛋白质组学方法对于肥胖症及糖尿病相关的多肽进行识别～作为潜在的识别分子和治疗靶象。 第二方面是用于植物生长发育及其调控机理研究。其主要研究的内容包括以下几个方面: ,1,据于植物生长发育相关的蛋白质组学研究～如与植物光周期相关蛋白质组研究。 ,2,植物感应环境胁迫后细胞内蛋白质组谱的变化 ,3,种子发育过程中特异蛋白质的表达及其功能分析 ,4,信号识别、转导途径中蛋白组分分析 ,5,细胞器蛋白质组 蛋白质组学研究尚处于一个起始阶段～我国在这方面的研究工作也才起步。目前国内许多大学和研究院所正在组织人员准备开展这方面的工作～希望在这方面有一席之地～但据了解～许多单位尚为建立一个合适的实验体系. Chapte 13 Protein Synthesis, Targeting, and Turnover 13.1 The cellular machinery of protein synthesis 13.1.1 Messenger RNA is the template for protein synthesis 13.1.2 Transfer RNAs order activated amino acids on the mRNA template 13.1.4 Ribosomes are the site of protein synthesis 13.2 The Genetic code 13.2.1 The code was deciphered with the help of synthetic messengers 13.2.2 The code is highly degenerate 13.2.3 Wobble introduces ambiguity into codon-anticodon interactions 13.2.4 The code is not universal 13.2.5 The rules regarding codon-anticodon pairing are species-specific 13.3 The Steps in translation 13.3.1