STRUCTURE/STABILITY OF AN EXTREME THERMOPHILE PROTEIN

这个项目的设计是为了描述 一种DNA结合蛋白，来自超嗜热性Sulfolobus。重点是 在稳定性和功能上都是如此。来自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 用于折叠和在高温下工作(通过80至110度 c)。人们对超嗜热蛋白的结构知之甚少，而且 对它们的折叠和折叠的热力学所知更少 功能。7kD染色质蛋白Sac7d和Sso7d是为数不多的 到目前为止所研究的可逆展开的超嗜热性蛋白质， 允许进行详细的热力学研究。它们还提供了一种简单的 研究非特异性DNA结合热力学的模型系统 使用为此目的而设计的蛋白质。当前关于 非特异性蛋白质-DNA相互作用的热力学在很大程度上是基于 序列特异性蛋白与非共识结合的研究进展 序列。 分子稳定自由能的首次测定 在这个项目的初始阶段进行了超嗜热蛋白的研究。 利用重组Sac7d。这项工作现在将扩展到本机 蛋白质，以及对折叠热力学的广泛研究 定点突变。特别值得关注的将是一个 一种平移后结构的表征和热力学研究 修饰，可能是赖氨酸甲基化，这增加了Sac7d 稳定性。这种修饰的重要性，表面离子相互作用， 离子结合和堆芯填充将通过以下组合进行研究 定点突变，核磁共振，化学变性，扫描和 滴定量热法。 Sac7d和Sso7d作为单体非特异性地结合双链DNA。这 是一种特别简单的详细结构热力学系统 个体相互作用和非特定分子的热力学研究 有约束力的。DNA中离子和非离子相互作用的重要性 结合将通过扫描和滴定量热法进行研究 使用荧光和圆二向色性以及特定位置的测量 诱变。 从这个项目中获得的信息将有助于我们有能力 设计和控制蛋白质稳定性和蛋白质-DNA相互作用 在医学和生物技术方面的潜在应用。

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.