CRYSTALLOGRAPHIC STUDY OF GLASS-TRANSITION IN HYPERTHERMOPHILIC ISOPROPYLMALATE

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 已知蛋白质动力学在蛋白质功能中起重要作用。 正常功能需要一定水平的内部流动性，当蛋白质经历所谓的玻璃化转变时，它们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~ 180 K，这与结构和计算数据一起导致了它由水合壳控制的假设，即蛋白质动力学受溶剂的奴役。 我们对极端嗜热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到~ 250 K。 拟议的实验包括仔细检查IPMDH中的蛋白质动力学在原子分辨率在不同的温度下，以解开超嗜热酶的玻璃化转变的分子机制。 此外，我们感兴趣的是使用类似的多温度数据收集策略，在原子分辨率，以研究蛋白质无序的温度依赖性在一个流产的三元复合物的E。大肠杆菌二氢叶酸还原酶（DHFR）与NADP+和叶酸结合。之前在100 K下从这些晶体收集的数据延伸到0.95 º以上，表明蛋白质的几个区域是无序的，并且这种无序可能与之前提出的催化相关蛋白质动力学有关。 然而，建立这种障碍的功能相关性需要衍射数据收集的温度下，酶可以催化氢化物转移。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.