Time-resolved analysis of enzyme reaction mechanism by Laue Crystallography.

通过劳厄晶体学对酶反应机制进行时间分辨分析。

• 批准号: 09680593
• 负责人: KATAYANAGI Katsuo
• 金额: $ 2.11万
• 依托单位: HIROSHIMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680593/
• 关键词: Time resolved Laue crystallography; X-ray structure; flexibility of protein; mutant protein; white X-ray; compressibility; DHFR; dynamic structure; X線結晶構造解析

The dynamic property of protein is quite important as well as its 3D structure for understanding its function. Therefore, we attempted to clarify the protein dynamics by means of X-ray structural analyses, focusing on B.ccli dihydrofolate reductase (DHFR). As the crystallization condition of DHFR was not registered on BMCD database and the method by Kraut's group was not reproducible, we consumed long time for its crystallization. First, we examined the stability of the obtained crystal for exposing the white X-ray in the time-resolved Laue experiment. In general, protein crystals decay by the white X-ray which contains wide range of wavelength, but we could obtain an excellent Laue pattern which is suitable enough for the dynamic studies. The Laue dynamic study requires not only X-ray crystallography but also the control of chemical reactions and physical characterization. Then, we studied the structure-flexibility relationship of DHFR before the Laue experiment. Vie made some DHFR mu … More tants at site 67, which locates on one of the flexible loops, and determined their crystal structure. On the other hand, we measured the adiabatic compressibility of the mutants at sites 67, 121, and 145, which is dominantly determined by the internal cavity in the protein molecule. Interestingly, we could find out the good correlation between the compressibility and the cavity volume derived from the crystal structures. Moreover, the distribution of the cavities in the protein molecule were found to be quite different with the mutants. On the other hand, there was no additivity in the function and stability of double mutants at sites 121 and 67 which separates about 28 A, suggesting the existence of long-distance interaction. Thus, the difference in the cavity distribution of mutants can be explained by such long-distance effects. As temperature is quite important factor for the flexibility, we applied cryo-X-ray measurement for G67A mutant at 130 and 190K.The B-factor decreased by cooling, but the extent was quite site-specific. Especially, the loop regions on the molecular surface and helix alpha C, which participates in the substrate binding, showed quite large decrease in flexibility. These structure and flexibility relationships will present important information for planning the Laue experiments. Less

蛋白质的动态特性及其三维结构对于理解其功能非常重要。因此，我们试图通过x射线结构分析来澄清蛋白质动力学，重点是B.ccli二氢叶酸还原酶（DHFR）。由于DHFR结晶条件未在BMCD数据库中登记，且Kraut等人的方法不具有重复性，结晶时间较长。首先，我们在时间分辨劳厄实验中检测了所获得的晶体在暴露白色x射线时的稳定性。一般来说，蛋白质晶体是通过波长范围很宽的白x射线衰减的，但我们可以得到一个很好的劳厄图，它足够适合于动态研究。劳厄动力学研究不仅需要x射线晶体学，还需要化学反应的控制和物理表征。然后，在Laue实验之前，我们研究了DHFR的结构-柔性关系。Vie在位于其中一个柔性环上的位点67上制备了一些DHFR，并确定了它们的晶体结构。另一方面，我们测量了突变体在位点67、121和145的绝热压缩性，这主要是由蛋白质分子的内部空腔决定的。有趣的是，我们可以从晶体结构中发现压缩率与空腔体积之间的良好相关性。此外，发现突变体在蛋白质分子中的空腔分布有很大的不同。另一方面，双突变体的功能和稳定性在相隔约28 A的121和67位点上没有可加性，表明存在远距离相互作用。因此，突变体在腔内分布的差异可以用这种远距离效应来解释。由于温度是影响柔性的重要因素，我们对G67A突变体在130和190K下进行了低温x射线测量。b因子随降温而降低，但程度有明显的地点特异性。特别是分子表面的环区和参与底物结合的螺旋α C，柔韧性下降较大。这些结构和灵活性关系将为规划劳厄实验提供重要信息。少