STRUCTURE/STABILITY OF AN EXTREME THERMOPHILE PROTEIN

作为极端的结构和稳定性的长期研究的一部分， 嗜热蛋白，我们提出了一个彻底的，定量研究的 极端耐热Sac 7 d的结构、稳定性和DNA结合 嗜热硫化叶菌（Sulfolobus acidocaldarius）的蛋白质， 92 C. 极端嗜热蛋白是高度优化的系统， 预期包含对高效合理蛋白质有用的信息 医学工程和生物技术。 Sac 7 d蛋白提供 一个顺从的，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 其可逆地解折叠并且缺乏二硫键和辅因子。 初步数据（NMR、DSC、部分比容）表明， 蛋白质折叠具有显著的二级结构（a-螺旋和五个螺旋）， B-片的股线）和填充良好的芯。 该项目将分为 分为三个部分： 首先，NMR将用于获得野生的高分辨率结构 型蛋白质在溶液中使用距离几何，限制分子 动力学和全松弛矩阵细化。 除了 传统的方法，一个新的蒙特卡罗方法将用于定义 NMR结构的精确度和准确度。 的可靠性 刚性模型将通过比较 拟合NOESY数据的标准误差。 不精确性与 关于刚性模型将作为一个可能的措施进行研究， 灵活性. 慢交换核的氢交换动力学 将测定蛋白质的酰胺氢并与那些 获得良好表征的嗜温蛋白以及Sac 7 d 突变体蛋白 第三，将结合DSC和NMR的定点诱变进行研究。 用来探测蛋白质的稳定性。 除了具体 NMR结构表明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 研究了 有待检验的操作假设是， 填料和最佳货车德瓦尔斯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。 的 核心的最佳包装的贡献将调查， 使用定点截短亮氨酸、异亮氨酸和缬氨酸残基 诱变 折叠自由能的变化 将去除的甲基/亚甲基/次甲基与观察到的进行比较 在嗜温蛋白质中。 蛋白质的堆积密度将是 使用Richard的Voronoi多面体方法计算。 置信限 对于基于NMR结构计算的堆积密度， 使用Monte Carlo精确度确定。 此外，部分 测量野生型和突变蛋白的比容。

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.