STRUCTURE/STABILITY OF AN EXTREME THERMOPHILE PROTEIN

作为对Extreme结构和稳定性的长期研究的一部分 嗜热蛋白，我们建议对其进行彻底的、定量的研究 极耐高温Sac7d的结构、稳定性及其与DNA的结合 来自Sulfolobus acidocaldarius的蛋白质，这是一种嗜热菌，可生长到 92C。极端嗜热蛋白是高度优化的系统， 预期包含对有效的理性蛋白质有用的信息 医学和生物技术的工程学。Sac7d蛋白提供 一种顺从的、行为良好的紧凑系统，用于探测物理基础 蛋白质的稳定性。它是已知的最小、最稳定的蛋白质 其可逆地展开，并且缺乏二硫键和辅因子。 初步数据(核磁共振、差示扫描量热、部分比体积)表明 蛋白质折叠具有显著的二级结构(一个a-螺旋和五个 B-Sheet链)和包装良好的核心。该项目将被划分为 分成三个部分： 首先，将使用核磁共振获得野生动物的高分辨率结构 利用距离几何和受限分子对溶液中的蛋白质进行分类 动力学，以及一个完全松弛的矩阵精化。除了……之外 传统方法中，将使用一种新的蒙特卡罗方法来确定 核磁共振结构的精密度和准确性。一台计算机的可靠性 刚性模型将通过比较标准偏差进行测试 对NOESY数据的标准误差进行拟合。的不精确度 关于刚性模型的研究将作为一种可能的测量方法 灵活性。慢换芯的氢交换动力学 蛋白质的酰胺氢将被测定，并与 获得了具有良好特性的中亲蛋白以及Sac7d 突变蛋白质。 第三，结合DSC和核磁共振的定点突变将是 用来探测蛋白质的稳定性。除了具体的 由核磁共振结构指示的相互作用可能是重要的 在稳定蛋白质方面，堆积密度的贡献将是 调查过了。需要检验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的因素 增强了极端嗜热蛋白质的稳定性。这个 将通过以下方式调查堆芯最佳填充的贡献 利用定点定位技术截断亮氨酸、异亮氨酸和缬氨酸残基 诱变。折叠机自由能的变化 将去除的甲基/亚甲基/亚甲基团与观察到的基团进行比较 在中亲蛋白质中。蛋白质的堆积密度将是 用Richard的Voronoi多面体方法计算。置信限 对于基于核磁共振结构计算的堆积密度将为 使用蒙特卡罗精确度确定。此外，部分 将测量野生型和突变型蛋白的比容。

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.