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.

高疗蛋白在结构生物学方面特别感兴趣，因为它们旨在在寿命的高温下（即80至110摄氏度）发挥作用。这项工作的长期目标是利用高疗蛋白的独特属性来增强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，而且对于扩展可用于研究蛋白质过程（例如折叠和结合）的实验范围。在这项工作中，我们建议利用两种高疗蛋白的高热和酸稳定性，以定量描述离子相互作用在折叠中的重要性以及在蛋白-DNA结合中扭曲DNA的能量成本。现在可以使用多种热疗蛋白的结构，并且该数据已用于提出有理解释，以增强这些蛋白质在高温下的稳定性和功能。最常见的解释是更多的离子对，预计在高温下将越来越重要。对于这些蛋白质中提出的离子对相互作用的相对重要性的深入热力学研究很少，并且没有对预测温度依赖性的实验证明。 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高疗蛋白。鉴于高密度的离子对以及我们在广泛条件下定义这些蛋白质稳定性的能力，它们非常适合研究高疗法蛋白质离子相互作用的重要性和行为。 SAC7D和SSO7D还提供了相对简单的模型系统，用于研究蛋白-DNA结合的能量学，因为它们诱导了任何蛋白质观察到的DNA中最大的扭结之一。高热稳定性将在此使用来定义DNA结合和弯曲的能量，以可靠地分离折叠，结合和弯曲的效果。这些相互作用将通过扫描和滴定量热法，NMR，定向诱变，圆形二色性和小角度X-射线散射的组合进行研究。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，并在医学和生物技术中的潜在应用。