CRYSTALLOGRAPHIC STUDY OF GLASS-TRANSITION IN HYPERTHERMOPHILIC ISOPROPYLMALATE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 蛋白质动力学在蛋白质功能中起着重要作用。正常的功能需要一定程度的内部流动性，当蛋白质经历所谓的玻璃化转变时，它们就失去了结合底物的能力。玻璃化转变在许多蛋白质中普遍发生在~180K，结合结构和计算数据，提出了玻璃化转变受水化壳层控制的假说，即蛋白质动力学受制于水化壳。通过溶剂法。我们对超高温异丙基苹果酸脱氢酶(IPMDH)在不同温度下的蛋白质动力学的初步研究表明，该蛋白质的玻璃化转变移动到~250K。该实验包括在不同温度下以原子分辨率仔细研究IPMDH中的蛋白质动力学，以揭示超高温酶玻璃化转变的分子机制。此外，我们有兴趣使用类似的原子分辨率多温度数据收集策略来研究大肠杆菌二氢叶酸还原酶(DHFR)与NADP+和叶酸结合的流产三元复合体中蛋白质无序的温度依赖性。以前从这些晶体中收集的100K数据延伸到0.95°以上，并表明蛋白质的几个区域是无序的，这种无序可能与先前提出的催化相关的蛋白质动力学有关。然而，要确定这种紊乱的功能相关性，需要在酶能够催化氢化物转移的温度下收集衍射数据。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Protein dynamics is known to play an important role in protein function. Certain level of internal mobility is required for proper function, and when proteins undergo so-called glass-transition, they lose the ability to bind substrates. Glass transition occurs universally at ~180K in many proteins, which together with the structural and computational data led to hypotheses that it is controlled by hydration shell, i.e. that protein dynamics is ?enslaved? by solvent. Our preliminary study of protein dynamics in hyperthermophilic isopropylmalate dehydrogenase (IPMDH) at different temperatures indicates that glass-transition for this protein is shifted to ~250K. The proposed experiment includes careful examination of protein dynamics in IPMDH at atomic resolution at different temperatures in order to unravel the molecular mechanism of glass-transition in hyperthermophilic enzymes. Furthermore, we are interested in using a similar multi-temperature data collection strategy at atomic resolution to investigate the temperature dependence of protein disorder in an abortive ternary complex of E. coli dihydrofolate reductase (DHFR) bound to NADP+ and folate. Previous data collected from these crystals at 100 K extends to beyond 0.95 ¿ and indicates that several regions of the protein are disordered and that this disorder may be related to previously proposed catalytically relevant protein dynamics. However, establishing the functional relevance of this disorder requires diffraction data collected at a temperature at which the enzyme can catalyze hydride transfer.