Nakasako M
Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Corporation (JST), Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi 1-1-1, Bunkyoku, Tokyo, Japan.
J Mol Biol. 1999 Jun 11;289(3):547-64. doi: 10.1006/jmbi.1999.2795.
The hydration structure of bovine beta-trypsin was investigated in cryogenic X-ray diffraction experiments. Three crystal forms of the enzyme inhibited by benzamidine with different molecular packing were selected to deduce the hydration structure for the entire surface of the enzyme. The crystal structures in all three of the crystal forms were refined at the resolution of 1.8 A at 100 K and 293 K. The number of hydration water molecules around the enzyme at 100 K was 1.5 to two times larger than that at 293 K, indicating that the motion of hydration water was quenched by cooling. In particular, the increase in the number of hydration water molecules was prominent on flat and electrostatically neutral surface areas. The water-to-protein mass ratio and the radius of gyration of a structural model of hydrated trypsin at 100 K was consistent with the results obtained by other experimental techniques for proteins in solution. Hydration water molecules formed aggregates of various shapes and dimensions, and some of the aggregates even covered hydrophobic residues by forming oligomeric arrangements. In addition, the aggregates brought about large-scale networks of hydrogen bonds. The networks covered a large proportion of the surface of trypsin like a patchwork, and mechanically linked several secondary structures of the enzyme. By merging the hydration structures of the three crystal forms at 100 K, a distribution function of hydration water molecules was introduced to approximate the static hydration structure of trypsin in solution. The function showed that the negatively charged active site of trypsin tended to be easily exposed to bulk solvent. This result is of interest with respect to the solvent shielding effect and the recognition of a positively charged substrate by trypsin.
在低温X射线衍射实验中研究了牛β-胰蛋白酶的水化结构。选择了三种被苯甲脒抑制且具有不同分子堆积方式的酶晶体形式，以推断该酶整个表面的水化结构。在100 K和293 K下，以1.8 Å的分辨率对所有三种晶体形式的晶体结构进行了精修。100 K时酶周围的水化水分子数量比293 K时大1.5至两倍，这表明冷却使水化水的运动受到抑制。特别是，在平坦且静电中性的表面区域，水化水分子数量的增加尤为显著。100 K时水化胰蛋白酶结构模型的水与蛋白质质量比及回转半径与其他蛋白质溶液实验技术所得结果一致。水化水分子形成了各种形状和尺寸的聚集体，其中一些聚集体甚至通过形成寡聚排列覆盖了疏水残基。此外，这些聚集体形成了大规模的氢键网络。这些网络像拼布一样覆盖了胰蛋白酶表面的很大一部分，并在机械上连接了该酶的几个二级结构。通过合并100 K时三种晶体形式的水化结构，引入了水化水分子的分布函数来近似胰蛋白酶在溶液中的静态水化结构。该函数表明，胰蛋白酶带负电荷的活性位点倾向于容易暴露于本体溶剂中。这一结果对于溶剂屏蔽效应以及胰蛋白酶对带正电荷底物的识别具有重要意义。
