SINGLE PROTON EXCHANGE KINETICS IN PROTEINS

我们的具体目标是双重的，继续我们的研究机制。 蛋白质氢交换，并确定它们之间的关系 不同亚分子的内迁移率和热力学稳定性 BPTI的结构域。这些是高度相关的；来自每个领域的结论 对研究结果的解释有直接关系。 其他的。蛋白质中的氢交换是指同型交换 蛋白质氨氢与溶剂氢的反应动力学。汇率 是内部蛋白质运动的函数，它使交换的NH暴露于 溶剂型。从历史上看，氢交换提供了第一个实验 洞察蛋白质结构的动力学方面。我们测量 用~1H核磁共振研究指定的NH质子的交换率。我们的实验室已经生产出一种 对基本理解的一些重大贡献 蛋白质中氢交换的机制。我们建议继续这样做 查询大道，使用模型蛋白牛胰腺胰酶 抑制剂(BPTI)。此外，我们正在探索两者之间的关系 蛋白质动态结构和蛋白质热力学稳定性。 1)蛋白质中的氢交换机制涉及两个中心 问题。年汇率放缓的结构性因素是什么？ 蛋白质与同等的延伸肽相比？什么类型的 构象波动导致埋藏的NH基团暴露在 当蛋白质处于折叠状态时是溶剂吗？我们研究了这些问题 通过测定交换反应的基本动力学，即 酸碱交换速率常数及其随时间的变化 BPTI在溶液和晶体中的温度、pH和离子强度。 我们使用通过定点突变获得的BPTI变异体来探测 蛋白质氢交换的基本化学和构象机制。 2)蛋白质内部迁移率与 热力学稳定性？亚分子堆积结构域，或微结构域， 都是基于氢交换性质定义的。这些是 “结”、“矩阵”和“面”。结是坚硬的疏水核心 交换质子最慢的原子。矩阵是 由埋藏的内部更灵活的区域组成，包含 更快地交换埋藏的NH。我们发现，BPTI中的基质有两个 组件，a)由长的、重叠的非周期的矩阵环组成 包裹埋藏水分子的节段，以及b)之间的铰链区 核心和母体。表面包含暴露在 溶剂，而不是分子内氢键。我们使用站点定向 选择性地扰乱BPTI的核心、基质或表面的突变。 然后我们通过氢交换测量内部灵活性的变化 蛋白水解性敏感性。合作社的热力学 变性由量热法和圆二色谱确定。时间 突变的BPTI的平均结构使用核磁共振距离和 受约束的分子动力学。通过这些方法，我们询问了一些 关于蛋白质动力学和蛋白质动力学之间关系的问题 热力学。

Our specific aims are twofold, to continue our studies on the mechanism of protein hydrogen exchange, and to determine the relationship between internal mobility and thermodynamic stability of various submolecular domains of BPTI. These are highly interrelated; conclusions from each area of study have a direct bearing on the interpretation of results from the other. Hydrogen exchange in proteins refers to the isotype exchange kinetics of a protein NH hydrogen with solvent hydrogen. The exchange rate is a function of internal protein motions that expose the exchanging NH to solvent. Historically, hydrogen exchange provided the first experimental insight into the dynamical aspect of protein structure. We measure exchange rates of assigned NH protons by 1H NMR. OUr lab has produced a number of significant contributions to the basic understanding of the mechanism of hydrogen exchange in proteins. We propose to continue this avenue of inquiry, using the model protein bovine pancreatic trypsin inhibitor (BPTI). In addition, we are probing the relationship between protein dynamic structure and protein thermodynamic stability. 1) The mechanism of hydrogen exchange in proteins involves two central question. What structural factors account for slowing of exchange rates in proteins compared to equivalent extended peptides? What kinds of conformational fluctuations lead to the exposure of buried NH groups to solvent when the protein is in the folded state? We study these questions by determining the basic kinetics of the exchange reaction, namely, the acid and base exchange rate constants, and their variation with temperature, pH and ionic strength for BPTI in solution and in crystals. We use BPTI variants obtained by site directed mutagenesis to probe the basic chemical and conformational mechanism of protein hydrogen exchange. 2) What is the relationship between protein internal mobility and thermodynamic stability? Submolecular packing domains, or microdomains, have been defined based on hydrogen exchange properties. These are the "knot", "matrix" and "surface". The knot is the rigid hydrophobic core of atoms that pack the very slowest exchanging protons. The matrix is composed of the more flexible regions of the buried interior containing more rapidly exchanging buried NH's. The matrix in BPTI, we find, had two components, a) the matrix loops comprised of long, overlapping aperiodic segments enclosing buried water molecules, and b) a hinge region between the core and the matrix. The surface contains the NH's that are exposed to solvent and not intramolecularly H-bonded. We use site-directed mutagenesis to selectively perturb the core, matrix, or surface of BPTI. Then we measure changes in internal flexibility by hydrogen exchange and proteolytic susceptibility. The thermodynamics of the cooperative denaturation are determined by calorimetry and circular dichroism. Time average structures of mutant BPTI's are modeled using NMr distances and constrained molecular dynamics. With these approaches we ask a number of questions regarding the relationship between protein dynamics and thermodynamics.