Dynamics-stability relationship in cytochromes c

描述(申请者提供)：中国高稳定性蛋白质的研究 在高温下茁壮成长的有机体(嗜热菌)是 阐明控制蛋白质稳定性和折叠的因素。尽管 二十多年来对嗜热菌蛋白质的研究，进化 高温适应背后的策略仍然是个谜。一个假设 已经出现的情况是，来自嗜热菌的蛋白质表现出增强 室温下的构象刚性(其中它们处于 “低温生物学”状态)与来自中温生物的同源蛋白质进行比较 (适应20-40摄氏度的生物)。相比之下，最近的计算研究 提示一些高度耐热的蛋白质具有增强的灵活性。 短时标(ps-ns)。这种短时间比例尺的灵活性旨在 通过增加折叠状态的熵来提高蛋白质的稳定性。 尽管人们对蛋白质之间的关系非常感兴趣 稳定性和动力学，来自有限数量的蛋白质的动力学 到目前为止，人们一直在研究嗜热菌。具体地说，几乎没有 短时间尺度运动的研究(小于微观的S)。目前，没有 一种结构上同系分子动力学的核磁共振表征 在广泛的时间范围内(PS-MS和 更长时间)已被报告。显然，需要更多的数据来了解 柔韧性和热稳定性是相关的。 这项研究的长期目标是从根本上洞察 蛋白质动力学与稳定性的关系。我们的指导性假设是 这种自然界使用以下策略来稳定蛋白质 嗜热菌：1)局部抑制。次全局构象和全局构象 在长时间(大于我们)的时间尺度上，整个蛋白质的波动。2) 允许更大的局部构象动力学(小于关于我们) 时间尺度。我们将通过描述两个分子的动力学特征来检验这一假设 结构上具有不同稳定性的同源细胞色素c(一种来自 一个来自嗜热菌，一个来自中温菌)和稳定性改变的突变体。 氢交换研究将被用来表征长时间尺度 动力学(US-ms)和15N松弛研究将揭示短时间尺度 动力学(ps-ns和更长)。氢交换数据还将用于 确定这些细胞色素结构单元的亚全局稳定性。 该项目的成功将提供有关 两种蛋白质稳定性与动力学的关系研究 类，并使我们更接近设计高度稳定的蛋白质的目标 变种人。

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.