蛋白质结晶之Tips X 氙气

蛋白质结晶之Tips X 氙气 X代表氙气(Xenon) 氙气可以结合到生物大分子特定的位点。氙气与蛋白形成的复合物可以用来做同型置换。蛋 白晶体与氙气在高压下反应即可得到蛋白晶体的重原子衍生物。大多数情况下，由于晶体和 母液在一个充满氙气的盒子中，所以不用调节母液来选择浸泡的条件。使用氙气的另外一个 优势是氙气与蛋白的结合较弱，所以不会太多影响蛋白的结构。通过调节气压，还能改变氙 气与蛋白结合的数量及位点，所以当常规的浸泡重原子的方法失败以后，可以尝试用氙气。 氙气与蛋白的结合是可逆的，所以当晶体的量很少时，用过的晶体还能再泡重原子。 氙气与蛋白之间的相互作用比较弱，不涉及静电相互作用。所以相比较重原子而言，氙气可 以结合到蛋白上很多位点。并且由于氙气与蛋白相互作用较弱，它不太可能改变蛋白晶体中 蛋白分子与蛋白分子之间的结合方式。用氙气得到的衍生物与蛋白晶体真实构象没有多大差 别。 氙气对母液的pH和离子强度没有明显影响。由于氙气与蛋白相互作用较弱，所以在泡制时需 要高压，以保证氙气充分结合到蛋白上各个可能结合的位点。此时，冷却处理就不会改变蛋 白与氙气的结合了。 利用氙气得到晶体衍生物的实验可以在常温下操作，数据收集也可以在特殊的装置中进行。 氙气的另外一个用途是可以用来做膜蛋白结构上不规则区域的成像。 原文: Xenon is a noble gas that binds to specific sites in a macromolecule. Xenon-protein complexes can often serve as heavy atom complexes for MIR structure determination. Heavy atom derivatives of protein crystals can be produced by pressurizing native crystals with xenon gas. In the vast majority of cases, the modification of the mother liquor to determine soaking conditions is avoided since the crystal and mother liquor are simply placed in a chamber pressurized with xenon gas. Another advantage of working with xenon is that it interacts weakly with a protein so isomorphism of the derivative with the native crystal is high. Also, the number of binding sites as well as the binding occupancies can often be changed by altering the pressure of the xenon gas. Xenon binding sites often differ from heavy metal binding sites so it can be useful to try xenon when traditional heavy atom soaks fail. Xenon binding is often reversible so if one has very few crystals, the same crystal could be used for heavy atom soak. The interaction between xenon and the protein is typically confined to weak dispersion forces and does not involve electrostatic interactions. Therefore, xenon should bind to different locations of the protein compared to most other heavy atom compounds. The weakness of the interaction between xenon and the protein makes it unlikely that xenon will interfere with crystal contacts. Xenon derivatives usually show good isomorphicity with the native crystal, resulting in high phasing power. Xenon seems to have little or no effect on the pH or ionic strength of the mother liquor. The weakness of the interactions between xenon and proteins requires a significant xenon concentration in the crystal’s mother liquor to enforce sufficient occupation of a potential binding site so most uses of xenon reported in the literature make use of high pressure equipment for xenon derivatization. At this time is appears cryocooling does not alter the protein’s xenon binding properties. Xenon derivatization can be performed at room temperature and data collection with xenon derivatives can be performed at room temperature using specially made pressure cells and capillaries or at cryogenic temperatures using CryoLoops. Another application of xenon in protein crystals is the crystallographic imaging of disordered areas of lipids or detergents in crystals of membrane proteins. References for using xenon as a derivative: 1. Binding of Xenon to Sperm Whale Myoglobin. Schoenborn B.P.; Watson, H.C.; Kendrew, J.C. (1965). Nature, 207, 28-30. 2. Cavities in proteins: structure of a metmyoglobin-xenon complex solved to 1.9&Angring. Tilton, R.F.; Kuntz, L.D.; Pesko, G.A. (1984) Biochemistry 23. 2849-2857. 3. Using Xenon as a Heavy Atom for Determining Phases in Sperm Whale Metmyoglobin. Vitali, J.; Robbins, A.H.; Almo, S.C.; Tilton, R.F. (1991). Journal of Applied Crystallography, 24, 931-935. 4. On the Preparation and X-ray Data Collection of Isomorphous Xenon Derivatives. Schiltz, M.; Prange, T.; Fourme, R. (1994). Journal of Applied Crystallography, 27, 950-960. 5. Successful flash-cooling of xenon-derivatised myoglobin crystals. Soltis, S.M.; Stowell, M.H.B.; Wiener, M.C.; Philips, G.N.; Rees, D.C. (1997).Journal of Applied Crystallography, 30, 190-194. 6. Freeze-Trapping Isomorphous Xenon Derivatives of Protein Crystals. Sauer, O.; Schmidt, A.; Kratky, C. (1997). Journal of Applied Crystallography, 30, 476-486. 7. Protein Crystallography at Ultra-Short Wavelengths: Feasibility Study of Anomalous Dispersion Experiments at the Xenon K-Edge. Schiltz, M., Kvick, A., Svensson, O., Shepard, W., De LaFortelle, E., Prange, T., Kahn, R. & Fourme, R. (1997). Journal of Synchrotron Radiation , 4, 287-297. 8. A method to stabilize reduced and/or gas treated protein crystals by flash cooling under a controlled atmospher 9. Xavier Vermede et.al. J. App. Cryst, (1999) 32(3) 505-509 10. Better structures from better data through better methods: a review of developments in de novo macromolecular phasing techniques and associated instrumentation at LURE. Fourme, R. et al (1999) J. Synch. Rad. 6(4) 834-844 11. Timmins, P., Pebay-Peroula, E. & Welte, W. (1994). Biophys. Chem. 53, 27-36.