STRUCTURAL THEMODYNAMICS OF A HYPERTHERMOPHILE

超嗜热分子的结构热力学

• 批准号: 6636088
• 负责人: JOHN W SHRIVER
• 金额: $ 26.72万
• 依托单位: UNIVERSITY OF ALABAMA IN HUNTSVILLE
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/6636088
• 关键词: Archaea; DNA; DNA binding protein; X ray crystallography; bacterial proteins; binding sites; calorimetry; chemical stability; chromatin; circular dichroism; hydropathy; intermolecular interaction; ionic bond; model design /development; nuclear magnetic resonance spectroscopy; nucleic acid structure; osmotic pressure; physical model; protein denaturation; protein folding; protein structure function; site directed mutagenesis; structural biology; temperature; thermodynamics; thermophilic organism

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具有特殊的意义，因为它们被设计为在生命的温度上限（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，并提高我们在蛋白质工程和生物技术方面的能力。这些蛋白质是重要的，不仅提供线索，如何控制稳定性在一个合理的方式，但也为扩大实验范围的条件下，可用于研究蛋白质的过程，如折叠和结合。在这项工作中，我们建议利用两个超嗜热蛋白质的高热和酸稳定性，定量地描述离子相互作用的重要性，在折叠和能量成本扭曲的DNA在蛋白质-DNA结合。一些超嗜热蛋白质的结构现在是可用的，这些数据已被用来提出合理的解释，提高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，预计在高温下会越来越重要。已经有一些深入的热力学研究的相对重要性，建议在这些蛋白质中的离子对相互作用，并没有实验证明预测的温度依赖性。7 kD染色质蛋白Sac 7 d和Sso 7 d已被证明是用于稳定性研究的理想的超嗜热蛋白。鉴于高密度的离子对和我们在广泛的条件下定义这些蛋白质的稳定性的能力，它们是研究超嗜热蛋白质中离子相互作用的重要性和行为的理想选择。Sac 7 d和Sso 7 d也为研究蛋白质-DNA结合的能量学提供了相对简单的模型系统，因为它们诱导了对任何蛋白质观察到的最大的DNA扭结之一。这里将利用高热稳定性来定义在宽温度范围内DNA结合和弯曲的能量学，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、圆二色谱和小角X射线散射的组合进行研究。从这个项目中获得的信息将有助于我们设计和控制蛋白质稳定性和蛋白质-DNA相互作用的能力，以合理的方式在医学和生物技术中具有潜在的应用。