STRUCTURAL THEMODYNAMICS OF A HYPERTHERMOPHILE

超嗜热分子的结构热力学

基本信息

批准号：
6562683
负责人：
JOHN W SHRIVER
金额：
$ 24.71万
依托单位：
UNIVERSITY OF ALABAMA IN HUNTSVILLE
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-05-01 至 2005-04-30
项目状态：
已结题

项目摘要

Hyperthermophile proteins are of special interest in structural biology since they are designed to function at the upper temperature limit of life (viz. 80 to 110 degrees C). The long term goal of this work is to utilize the unique attributes of hyperthermophile proteins to increase our understanding of protein energetics and enhance our capabilities in protein engineering and biotechnology. These proteins are important not only for providing clues on how to control stability in a rational manner, but also for expanding the experimental range of conditions available for investigating protein processes such as folding and binding. In this work we propose to take advantage of the high thermal and acid stability of two hyperthermophile proteins to quantitatively describe the importance of ionic interactions in folding and the energetic cost of distorting DNA in protein-DNA binding. The structures of a number of hyperthermophile proteins are now available, and this data has been utilized to propose rational explanations for enhanced stability and function in these proteins at high temperature. The most common explanation is a greater number of ion pairs, which are expected to be increasingly important at high temperature. There have been few in-depth thermodynamic studies of the relative importance of the proposed ion pair interactions in these proteins and no experimental demonstration of the predicted temperature dependence. The 7 kD chromatin proteins Sac7d and Sso7d have proven to be ideal hyperthermophile proteins for studies of stability. Given a high density of ion pairs and our ability to define the stabilities of these proteins over a wide range of conditions, they are ideal for studies of the importance and behavior of ionic interactions in hyperthermophile proteins. Sac7d and Sso7d also provide relatively simple model systems for studies of the energetics of protein-DNA binding since they induce one of the largest kinks in DNA observed for any protein. The high thermal stability will be utilized here to define the energetics of DNA binding and bending over a wide temperature range to reliably separate the effects of folding, binding and bending. These interactions will be investigated by a combination of scanning and titration calorimetry, NMR, site-directed mutagenesis, circular dichroism, and small-angle X- ray scattering. Information gained from this project will contribute to our ability to design and control protein stability and protein-DNA interactions in a rational manner with potential applications in medicine and biotechnology.

超嗜热蛋白在结构生物学中特别受关注，因为它们被设计为在生命上限温度（即 80 至 110 摄氏度）下发挥作用。这项工作的长期目标是利用超嗜热蛋白的独特属性来增加我们对蛋白质能量学的理解并增强我们在蛋白质工程和生物技术方面的能力。这些蛋白质不仅对于提供如何以合理方式控制稳定性的线索很重要，而且对于扩大可用于研究蛋白质过程（例如折叠和结合）的实验条件范围也很重要。在这项工作中，我们建议利用两种超嗜热蛋白的高热稳定性和酸稳定性来定量描述折叠中离子相互作用的重要性以及蛋白质-DNA 结合中扭曲 DNA 的能量成本。现在，许多超嗜热蛋白的结构已经可用，并且这些数据已被用来对这些蛋白在高温下增强的稳定性和功能提出合理的解释。最常见的解释是离子对数量增多，预计这些离子对在高温下会变得越来越重要。对于这些蛋白质中所提出的离子对相互作用的相对重要性，几乎没有进行深入的热力学研究，也没有对预测的温度依赖性进行实验证明。 7 kD 染色质蛋白 Sac7d 和 Sso7d 已被证明是用于稳定性研究的理想超嗜热蛋白。鉴于离子对的高密度以及我们在各种条件下定义这些蛋白质稳定性的能力，它们非常适合研究超嗜热蛋白质中离子相互作用的重要性和行为。 Sac7d 和 Sso7d 还为研究蛋白质-DNA 结合的能量学提供了相对简单的模型系统，因为它们诱导了任何蛋白质中观察到的最大 DNA 扭结之一。这里将利用高热稳定性来定义 DNA 结合和弯曲在较宽温度范围内的能量学，以可靠地分离折叠、结合和弯曲的影响。这些相互作用将通过扫描和滴定量热法、NMR、定点诱变、圆二色性和小角 X 射线散射的组合来研究。从该项目中获得的信息将有助于我们以合理的方式设计和控制蛋白质稳定性和蛋白质-DNA 相互作用的能力，并在医学和生物技术方面具有潜在的应用。