Kadri A, Lorber B, Charron C, Robert M-C, Capelle B, Damak M, Jenner G, Giegé R
Département Mécanismes et Macromolécules de la Synthèse Protéique et Cristallogenèse, UPR 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS, 67084 Strasbourg CEDEX, France.
Acta Crystallogr D Biol Crystallogr. 2005 Jun;61(Pt 6):784-8. doi: 10.1107/S0907444905007109. Epub 2005 May 26.
Pressure is a non-invasive physical parameter that can be used to control and influence protein crystallization. It is also found that protein crystals of superior quality can be produced in gel. Here, a novel crystallization strategy combining hydrostatic pressure and agarose gel is described. Comparative experiments were conducted on hen and turkey egg-white lysozymes and the plant protein thaumatin. Crystals could be produced under up to 75-100 MPa (lysozymes) and 250 MPa (thaumatin). Several pressure-dependent parameters were determined, which included solubility and supersaturation of the proteins, number, size and morphology of the crystals, and the crystallization volume. Exploration of three-dimensional phase diagrams in which pH and pressure varied identified growth conditions where crystals had largest size and best morphology. As a general trend, nucleation and crystal-growth kinetics are altered and nucleation is always enhanced under pressure. Further, solubility of the lysozymes increases with pressure while that of thaumatin decreases. Likewise, changes in crystallization volumes at high and atmospheric pressure are opposite, being positive for the lysozymes and negative for thaumatin. Crystal quality was estimated by analysis of Bragg reflection profiles and X-ray topographs. While the quality of lysozyme crystals deteriorates as pressure increases, that of thaumatin crystals improves, with more homogeneous crystal morphology suggesting that pressure selectively dissociates ill-formed nuclei. Analysis of the thaumatin structure reveals a less hydrated solvent shell around the protein when pressure increases, with approximately 20% less ordered water molecules in crystals grown at 150 MPa when compared with those grown at atmospheric pressure (0.1 MPa). Noticeably, the altered water distribution is seen in depressurized crystals, indicating that pressure triggers a stable structural alteration on the protein surface while its polypeptide backbone remains essentially unaltered.
压力是一种非侵入性物理参数，可用于控制和影响蛋白质结晶。还发现可以在凝胶中制备出高质量的蛋白质晶体。在此，描述了一种结合静水压力和琼脂糖凝胶的新型结晶策略。对鸡和火鸡蛋清溶菌酶以及植物蛋白奇异果甜蛋白进行了对比实验。在高达75 - 100兆帕（溶菌酶）和250兆帕（奇异果甜蛋白）的压力下可以产生晶体。确定了几个与压力相关的参数，包括蛋白质的溶解度和过饱和度、晶体的数量、大小和形态以及结晶体积。探索了pH值和压力变化的三维相图，确定了晶体尺寸最大且形态最佳的生长条件。一般趋势是，成核和晶体生长动力学发生改变，并且在压力下成核总是增强。此外，溶菌酶的溶解度随压力增加而增加而奇异果甜蛋白的溶解度则降低。同样，高压和常压下结晶体积的变化相反，溶菌酶为正，奇异果甜蛋白为负。通过分析布拉格反射图谱和X射线形貌图来评估晶体质量。虽然溶菌酶晶体的质量随着压力增加而变差，但奇异果甜蛋白晶体的质量得到改善，更均匀的晶体形态表明压力选择性地解离了不良形成的核。对奇异果甜蛋白结构的分析表明，压力增加时蛋白质周围的溶剂化层水合程度降低，与在常压（0.1兆帕）下生长的晶体相比，在150兆帕下生长的晶体中有序水分子减少约20%。值得注意的是，在减压晶体中也观察到了水分布的改变，这表明压力在蛋白质表面引发了稳定的结构改变，而其多肽主链基本保持不变。
