STRUCTURE/STABILITY OF AN EXTREME THERMOPHILE PROTEIN

极端嗜热蛋白质的结构/稳定性

• 批准号: 2701595
• 负责人: JOHN W SHRIVER
• 金额: $ 19.23万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 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 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701595
• 关键词: Archaea; DNA binding protein; X ray crystallography; bacterial proteins; calorimetry; chemical models; circular dichroism; hydrogen bond; intermolecular interaction; nuclear magnetic resonance spectroscopy; protein denaturation; protein folding; protein structure; site directed mutagenesis; structural biology; thermodynamics; thermophilic organism; thermostability

This project is designed to characterize the structural thermodynamics of a DNA binding protein from the hyperthermophile Sulfolobus. The emphasis is on both stability and function. Proteins from hyperthermophiles are of interest in protein engineering and biotechnology since they are designed for both folding and functioning at high temperature (via 80 to 110 degrees C). Little is known about the structures of hyperthermophile proteins, and even less is known about the thermodynamics of both their folding and function. The 7 kD chromatin proteins Sac7d and Sso7d are two of the few hyperthermophile proteins studied to date which unfold reversibly, permitting detailed thermodynamic studies. They also provide a simple model system for studies of the thermodynamics of non-specific DNA-binding using proteins that are designed for that purpose. Current ideas on the thermodynamics of nonspecific protein-DNA interactions are largely based on studies of sequence-specific proteins binding to non-consensus sequences. The first determination of the free energy of stabilization of a hyperthermophile protein was performed in the initial phase of this project using recombinant Sac7d. This work will now be extended to the native protein along with an extensive study of the folding thermodynamics with site-directed mutagenesis. Of particular interest will be a characterization and thermodynamic study of a post-translational modification, possibly lysine methylation, which increases the Sac7d stability. The importance of this modification, surface ionic interactions, ion binding, and core packing will be investigated by a combination of site-directed mutagenesis, NMR, chemical denaturation, and scanning and titration calorimetry. Sac7d and Sso7d bind double-stranded DNA non-specifically as monomers. This is an especially simple system for detailed structural thermodynamic studies of individual interactions and the thermodynamics of nonspecific binding. The importance of both ionic and nonionic interactions in DNA binding will be investigated by scanning and titration calorimetry, binding measurements using fluorescence and circular dichroism, and site-specific mutagenesis. Information gained from this project will contribute to our ability to design and control protein stability and protein-DNA interactions with potential applications in medicine and biotechnology.

该项目旨在表征的结构热力学 来自超嗜热菌硫叶菌的DNA结合蛋白。重点 是稳定性和功能。超嗜热菌的蛋白质是 对蛋白质工程和生物技术的兴趣，因为它们是设计 用于在高温（80至110度）下折叠和运行 C）。对超嗜热蛋白的结构知之甚少， 关于它们折叠的热力学， 功能7 kD染色质蛋白Sac 7 d和Sso 7 d是为数不多的 迄今为止研究的可逆解折叠的超嗜热蛋白， 允许详细的热力学研究。 他们还提供了一个简单的 DNA非特异性结合热力学研究的模型系统 使用专门为此目的设计的蛋白质。目前关于 非特异性蛋白质-DNA相互作用的热力学主要基于 序列特异性蛋白质与非共有蛋白质结合的研究 序列的 首次确定了一个稳定化的自由能， 超嗜热蛋白在本项目的初始阶段进行 使用重组Sac 7 d。这项工作现在将扩展到本机 蛋白质沿着与折叠热力学的广泛研究， 定点诱变。特别令人感兴趣的将是 一种翻译后酶的表征和热力学研究 修饰，可能是赖氨酸甲基化，这增加了Sac 7 d 稳定这种修饰的重要性，表面离子相互作用， 离子结合和核心包装将研究的组合， 定点诱变、NMR、化学变性和扫描， 滴定量热法 Sac 7 d和Sso 7 d作为单体非特异性地结合双链DNA。这 是一个特别简单的系统，详细的结构热力学 个体相互作用和非特异性热力学的研究 约束力DNA中离子和非离子相互作用的重要性 结合将通过扫描量热法和滴定量热法进行研究， 测量使用荧光和圆二色性，和位点特异性 诱变 从这个项目中获得的信息将有助于我们的能力， 设计和控制蛋白质稳定性和蛋白质-DNA相互作用， 在医学和生物技术中的潜在应用。