Dynamics-stability relationship in cytochromes c

细胞色素 c 的动力学稳定性关系

• 批准号: 7036499
• 负责人: KARA L. BREN
• 金额: $ 19.22万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7036499
• 关键词: chemical stability; chimeric proteins; circular dichroism; conformation; cytochrome c; electrospray ionization mass spectrometry; enzyme structure; molecular dynamics; nuclear magnetic resonance spectroscopy; protein sequence; thermodynamics; thermophilic organism; thermostability; ultraviolet spectrometry

DESCRIPTION (provided by applicant): Research on highly stable proteins from organisms that thrive at high temperatures (thermophiles) is integral to elucidating the factors that control protein stability and folding. Despite over two decades of research on proteins from thermophiles, the evolutionary strategies behind high-temperature adaptation remain enigmatic. One hypothesis that has emerged is that proteins from thermophiles display enhanced conformational rigidity at room temperature (in which they are in a "cryobiological" state) in comparison with homologous proteins from mesophiles (organisms adapted to 20 - 40 C). In contrast, recent computational studies suggest that some highly thermostable proteins have enhanced flexibility on short time scales (ps - ns). This short time scale flexibility is proposed to enhance protein stability by increasing the entropy of the folded state. Despite the high level of interest in the relationship between protein stability and dynamics, the dynamics of a limited number of proteins from thermophiles have been studied to date. In particular, there are few investigations of short time scale motions (less than micro s). Currently, no NMR characterization of the dynamics of a structurally homologous thermophile-mesophile pair through a wide range of time scales (ps - ms and longer) has been reported. Clearly, more data are needed to understand how flexibility and thermostability are related. The long-range goal of this research is to gain fundamental insight into the relationship between protein dynamics and stability. Our guiding hypothesis is that nature uses the following strategies for stabilizing proteins from thermophiles: 1) Suppression of local. subglobal and global conformational fluctuations throughout the protein on long (greater than us) time scales. 2) Allowing greater local conformational dynamics on short (less than about us) time scales. We will test this hypothesis by characterizing the dynamics of two structurally homologous cytochromes c with differing stabilities (one from a thermophile, and one from a mesophile) and mutants with altered stabilities. Hydrogen exchange studies will be employed to characterize long time scale dynamics (us -ms), and 15N relaxation studies will reveal short time scale dynamics (ps - ns, and longer). Hydrogen exchange data also will be used to determine subglobal stabilities of structural units of these cytochromes. Success in this project will provide fundamental information on the relationship between stability and dynamics of two proteins from a well-studied class and bring us closer to the goal of designing highly stable protein mutants.

描述（由申请人提供）：对来自 在高温下茁壮成长的生物（嗜热生物）是 阐明控制蛋白质稳定性和折叠的因素。尽管 经过二十多年对嗜热菌蛋白质的研究， 高温适应背后的策略仍然是个谜。一个假设 嗜热菌的蛋白质表现出增强的 在室温下的构象刚性（其中它们处于 “低温生物学”状态）与来自嗜温生物的同源蛋白质相比 （生物体适应20 - 40 ℃）。相比之下，最近的计算研究 这表明，一些高度热稳定的蛋白质具有增强的灵活性， 短时间尺度（ps - ns）。这种短时间尺度的灵活性是为了 通过增加折叠状态的熵来增强蛋白质的稳定性。 尽管人们对蛋白质与 稳定性和动力学，有限数量的蛋白质的动力学， 迄今为止已经对嗜热菌进行了研究。尤其是， 研究短时间尺度运动（小于微秒）。当前没有任何 一种结构上同系的动力学的NMR表征 通过宽范围的时间尺度（PS-MS和 更长）已被报道。显然，需要更多的数据来了解 柔性和热稳定性是相关的。 这项研究的长期目标是从根本上了解 蛋白质动力学与稳定性的关系我们的指导假设是 大自然使用以下策略来稳定蛋白质， 嗜热菌：1）抑制局部。亚全局和全局构象 整个蛋白质在长时间（大于我们）的时间尺度上的波动。（二） 允许更大的局部构象动力学短（小于约我们） 时间尺度我们将通过描述两种动力学来验证这一假设。 具有不同稳定性的结构同源的细胞色素c（一种来自a 嗜热菌和一种来自嗜温菌）和具有改变的稳定性的突变体。 氢交换研究将用于表征长时间尺度 动力学（us -ms）和15 N弛豫研究将揭示短时间尺度 动力学（ps - ns，以及更长）。氢交换数据也将用于 决定这些细胞色素结构单位的亚全局稳定性。 这一项目的成功将提供关于 经过充分研究的两种蛋白质的稳定性和动力学之间的关系 类，使我们更接近设计高度稳定的蛋白质的目标， 变种人