Juers D H, Matthews B W
Institute of Molecular Biology Howard Hughes Medical Institute and Department of Physics, 1229 University of Oregon, Eugene, OR 97403-1229, USA.
J Mol Biol. 2001 Aug 24;311(4):851-62. doi: 10.1006/jmbi.2001.4891.
Flash-freezing, which has become routine in macromolecular X-ray crystallography, causes the crystal to contract substantially. In the case of Escherichia coli beta-galactosidase the changes are reversible and are shown to be due to lattice repacking. On cooling, the area of the protein surface involved in lattice contacts increases by 50 %. There are substantial alterations in intermolecular contacts, these changes being dominated by the long, polar side-chains. For entropic reasons such side-chains, as well as surface solvent molecules, tend to be somewhat disordered at room temperature but can form extensive hydrogen-bonded networks on cooling. Low-temperature density measurements suggest that, at least in some cases, the beneficial effect of cryosolvents may be due to a density increase on vitrification which reduces the volume of bulk solvent that needs to be expelled from the crystal. Analysis of beta-galactosidase and several other proteins suggests that both intramolecular and intermolecular contact interfaces can be perturbed by cryocooling but that the changes tend to be more dramatic in the latter case. The temperature-dependence of the intermolecular interactions suggests that caution may be necessary in interpreting protein-protein and protein-nucleic acid interactions based on low-temperature crystal structures.
速冻在大分子X射线晶体学中已成为常规操作，它会使晶体大幅收缩。以大肠杆菌β-半乳糖苷酶为例，这种变化是可逆的，且已证明是由于晶格重新排列所致。冷却时，参与晶格接触的蛋白质表面面积增加50%。分子间接触有显著改变，这些变化主要由长的极性侧链主导。出于熵的原因，此类侧链以及表面溶剂分子在室温下往往有些无序，但在冷却时可形成广泛的氢键网络。低温密度测量表明，至少在某些情况下，低温保护剂的有益作用可能是由于玻璃化时密度增加，从而减少了需要从晶体中排出的大量溶剂的体积。对β-半乳糖苷酶和其他几种蛋白质的分析表明，分子内和分子间接触界面都可能因低温冷却而受到扰动，但后一种情况下的变化往往更为显著。分子间相互作用的温度依赖性表明，在基于低温晶体结构解释蛋白质-蛋白质和蛋白质-核酸相互作用时可能需要谨慎。
