STRUCTURE/STABILITY OF AN EXTREME THERMOPHILE PROTEIN

极端嗜热蛋白质的结构/稳定性

基本信息

批准号：
2187216
负责人：
JOHN W SHRIVER
金额：
$ 14.38万
依托单位：
SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
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 1997-04-30
项目状态：
已结题

项目摘要

As part of a long range study of structure and stability of extreme thermophile proteins, we propose a thorough, quantitative study of the structure, stability, and DNA-binding of the extremely thermostable Sac7d protein from Sulfolobus acidocaldarius, a thermophile which grows up to 92C. Extreme thermophile proteins are highly optimized systems that are expected to contain information useful for efficient rational protein engineering in medicine and biotechnology. The Sac7d protein provides an amenable, well-behaved compact system for probing the physical basis of protein stability. It is the smallest, most stable protein known which unfolds reversibly and lacks disulfide linkage and cofactors. Preliminary data (NMR, DSC, partial specific volume) indicate that the protein folds with significant secondary structure (an a-helix and five strands of B-sheet) and a well packed core. The project will be divided into three parts: First, NMR will be used to obtain a high resolution structure of the wild type protein in solution using distance geometry, restrained molecular dynamics, and a full-relaxation matrix refinement. In addition to traditional methods, a new Monte Carlo method will be used for defining the precision and accuracy of the NMR structure. The reliability of a rigid model will be tested by comparison of the standard deviation of the fit to the standard error of the NOESY data. The imprecision with respect to a rigid model will be investigated as a possible measure of flexibility. The hydrogen exchange kinetics of the slow exchanging core amide hydrogens of the protein will be determined and compared with those obtained for well characterized mesophile proteins as well as Sac7d mutants proteins. Third, site-directed mutagenesis in conjunction with DSC and NMR will be used to probe the stability of the protein. In addition to specific interactions indicated by the NMR structure to be potentially important in stabilizing the protein, the contribution of packing density will be investigated. The operating hypothesis to be tested is that efficient packing and optima van der Waals contacts can be an important factor in conferring enhanced stability on an extreme thermophile protein. The contribution of optimum packing of the core will be investigate by truncating leucine, isoleucine and valine residues using site directed mutagenesis. The change in free energy of folding per methyl/methylene/methine group removed will be compared to that observed in mesophile proteins. The packing density of the protein will be calculated using Richard's Voronoi polyhedra method. Confidence limits for the calculated packing density based on the NMR structure will be determined using the Monte Carlo precision. In addition, the partial specific volume of the wild type and mutant proteins will be measured.

作为极端结构和稳定性长期研究的一部分嗜热蛋白，我们建议对嗜热蛋白进行彻底的定量研究极其耐热的 Sac7d 的结构、稳定性和 DNA 结合来自酸热硫化叶菌的蛋白质，一种嗜热微生物，可生长至 92C。极端嗜热蛋白是高度优化的系统预计包含对有效理性蛋白质有用的信息医学和生物技术工程。 Sac7d 蛋白提供一个适合探测物理基础的、性能良好的紧凑系统蛋白质稳定性。它是已知最小、最稳定的蛋白质它可逆地展开并且缺乏二硫键和辅因子。初步数据（NMR、DSC、部分比容）表明具有显着二级结构（一个α螺旋和五个螺旋）的蛋白质折叠 B-sheet 股）和包装良好的核心。该项目将分为分为三部分：首先，核磁共振将用于获得野生的高分辨率结构使用距离几何、受限分子对溶液中的蛋白质进行类型分析动力学和全松弛矩阵细化。此外传统方法，将使用新的蒙特卡罗方法来定义 NMR 结构的精密度和准确度。的可靠性刚性模型将通过比较标准偏差进行测试符合 NOESY 数据的标准误差。不精确性关于刚性模型的研究将作为一种可能的衡量标准灵活性。慢速交换核的氢交换动力学将测定蛋白质的酰胺氢并与那些进行比较获得良好表征的嗜温蛋白以及 Sac7d 突变蛋白。第三，与 DSC 和 NMR 相结合的定点诱变将用于检测蛋白质的稳定性。除了具体的 NMR 结构表明相互作用具有潜在的重要意义在稳定蛋白质方面，堆积密度的贡献为调查了。要测试的操作假设是有效的包装和最佳范德华接触可能是一个重要因素赋予极端嗜热蛋白质增强的稳定性。这核心最佳封装的贡献将通过以下方式进行研究使用定点截断亮氨酸、异亮氨酸和缬氨酸残基诱变。每折叠自由能的变化除去的甲基/亚甲基/次甲基将与观察到的进行比较存在于嗜温蛋白质中。蛋白质的堆积密度为使用 Richard 的 Voronoi 多面体方法计算。置信限度基于 NMR 结构计算的堆积密度为使用蒙特卡罗精度确定。此外，部分将测量野生型和突变蛋白的比体积。