The Structural and Energetic Bases of Extreme Thermal Stability in Proteins

蛋白质极端热稳定性的结构和能量基础

• 批准号: 9604213
• 负责人: Frederick Dahlquist
• 金额: $ 24万
• 依托单位: University of Oregon Eugene
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-15 至 2000-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604213&HistoricalAwards=false
• 关键词: Structural; Energetic; Bases; Extreme; Thermal

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.

96-04213 Dahlquist本研究旨在阐明在95℃下经历可逆热解折叠的海洋热天牛Chey蛋白具有超热稳定性的本质。从枯草芽孢杆菌中分离到的Chey长度相同，氨基酸序列与TMY的同源性为75%，但变性温度降低了约35oC。这两种蛋白质中不同的30个残基中的每一个都将被取代，以监测每一个残基对蛋白质整体稳定性的贡献。选定的突变体将通过各种物理方法进行检测，包括圆二色谱、溶液核磁共振方法、差示扫描量热法和X射线结晶学，以确定蛋白质的热稳定性与其结构和动态性质之间的可能关系。还将描述TMY的热诱导结构变化，该变化发生在其热展开以下约50度。这种转变，类似于在来自超嗜热源的其他蛋白质中观察到的那样，导致TMY的15N-1H相关谱的广泛线宽和化学位移变化。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，以了解结构和能量对来自超嗜热源的蛋白质所见的特殊热稳定性的贡献。生活在很高温度下的生物体(嗜热菌)的蛋白质比生活在体温附近的生物体(中温菌)的类似蛋白质稳定得多。这项研究对一种行为良好的小蛋白Chey的稳定性来源进行了系统的研究，Chey参与了高温和中温生物的感觉信号传递。与来自中温来源的类似蛋白质相比，从超嗜热性HAS分离的这种蛋白质的大约20个氨基酸可能导致其热稳定性增加近50oC。这些残基将通过定点突变而发生变化，以便通过X射线衍射和核磁共振方法确定这些残基在蛋白质及其结构稳定性中的作用。此外，在超嗜热者的Chey蛋白中，随着温度的升高，在接近体温的情况下，结构也会发生变化。这种结构变化可能反映了在低温下从催化不活跃的形式向高温下的功能形式的变化。这一变化及其发生的原因将被描述为对蛋白质在极端环境条件下如何发挥作用的一般理解。