The Structural and Energetic Bases of Extreme Thermal Stability in Proteins

96-04213 Dahlquist 本研究旨在阐明海栖热袍菌（Thermotoga maritima，TMY）的CheY蛋白的超热稳定性，该蛋白在95 ℃时发生可逆的热去折叠。从枯草芽孢杆菌中分离的CheY与TMY长度相同，氨基酸序列有75%相同，但变性温度低约35 ℃。两种蛋白质中不同的30个残基中的每一个都将被取代，以监测每一个对蛋白质整体稳定性的贡献。选择的突变体将通过各种物理方法进行检查，包括圆二色性、溶液核磁共振方法、差示扫描量热法和X射线晶体学，以确定蛋白质的热稳定性与其结构和动力学性质之间的可能关系。 一个热诱导的结构变化TMY发生在其热展开以下约50度也将被表征。这种转变，类似于在其他蛋白质中观察到的超嗜热来源，导致广泛的线加宽和化学位移的变化，在15 N-1H相关光谱的TMY。 因此，在这项研究中获得的信息应该提供一个丰富的实验数据库的结构和能量的贡献，从超嗜热来源的蛋白质中看到的特殊的热稳定性的理解。 来自生活在非常高的温度下的生物（超嗜热生物）的蛋白质比来自生活在接近体温的生物（中温生物）的类似蛋白质稳定得多。这项研究进行了系统的调查来源的稳定性，在一个小的，表现良好的蛋白质，CheY，这是参与感觉信号在嗜热菌和嗜温菌。从嗜热菌中分离的这种蛋白质的约20个氨基酸可能是其热稳定性增加近50 ℃的原因，与来自嗜温来源的类似蛋白质相比。这些残基将通过定点突变而改变，从而可以识别这些残基在蛋白质稳定性及其结构中的作用，如通过X射线衍射和核磁共振方法所确定的。此外，随着温度的升高，来自超嗜热菌的CheY蛋白在接近体温时发生结构变化。这种结构变化可以反映从低温下的无催化活性形式到高温下的功能形式的变化。这种变化及其发生的原因将被表征，以产生对蛋白质在极端环境条件下如何发挥作用的一般理解。

96-04213 Dahlquist This study is designed to elucidate the nature of hyperthermostability in the CheY protein from Thermotoga maritima (TMY), which undergoes reversible thermal unfolding at 95oC. The CheY isolated from Bacillus subtilis is the same length and is 75% identical in amino acid sequence to TMY but denatures about 35oC lower in temperature. Each of the 30 residues that differ in the two proteins will be substituted in order to monitor the contribution that each makes to the overall stability of the protein. Selected mutants will be examined by a variety of physical methods including circular dichroism, solution nuclear magnetic resonance methods, differential scanning calorimetry and X- ray crystallography for possible relationships between the thermal stability of a protein and its structural and dynamic properties. A thermally-induced structural change in TMY that occurs about 50o below its thermal unfolding will also be characterized. This transition, similar to those observed in other proteins from hyperthermophilic sources, results in extensive line broadening and chemical shift changes in the 15N-1H correlation spectra of TMY. The information so gained in this study should provide a rich experimental database for the understanding of the structural and energetic contribution to the exceptional thermal stability seen in proteins from hyperthermophilic sources. Proteins from organisms that live at very high temperatures (hyperthermophiles) are much more stable than similar proteins from organisms that live near body temperature (mesophiles). This study undertakes a systematic investigation of the source of stability in a small, well behaved protein, CheY, which is involved in sensory signaling in both hyperthermophiles and mesophiles. About 20 amino acids of this protein, isolated from a hyperthermophile has, are likely to account for a near 50oC increase in its thermal stability, as compared to the similar protein from a mesophilic source. These residues will be varied by sitedirected mutation so that the role of these residues in the stability of the protein and its structure, as determined by X-ray diffraction and nuclear magnetic resonance methods, can be recognized. In addition, there is a change in structure as temperature increases that occurs near body temperature in the CheY protein from the hyperthermophile. This structural change may reflect a change from a catalytically inactive form at low temperature to a functional form at high temperature. This change and the reasons that it occurs will be characterized to yield a general understanding of how proteins function under extreme environmental conditions.