SINGLE PROTON EXCHANGE KINETICS IN PROTEINS

这笔赠款的长期目标是促进基础知识的发展 关于蛋白质氢交换的机制，一个独特而通用的探针 蛋白质的动态结构。蛋白质中的氢交换发生在 通过折叠态的内部运动，或通过全球合作 我们称之为交换的两个过程模型。晋级 理解每个过程，我们描述了它们之间的关系 折叠体的氢交换速率和平均(晶体)结构 国家，以及氢交换率和全球 正在展开。为此，由这笔赠款资助的工作自上一次 竞争更新产生了大量关于动态的数据， Small的8个变体的折叠热力学和晶体结构 模型蛋白，牛胰蛋白酶抑制物(BPTI)。这项研究 这份提案中的计划有两个；i)将我们建立的数据库 关于氢交换机理的进一步重要问题 蛋白质与氢交换测量的动力学关系 蛋白质平均结构(晶体或核磁共振)和蛋白质折叠，以及 Ii)检验我们将蛋白质动态结构与蛋白质联系起来的假说 折叠，即本态慢交换的核心是折叠 核心。具体目标包括以下几点。A)地面汇率 BPTI突变体中的NHS将被确定。B)双突变组合 我们将构建单点突变以检测BPTI 在低pH条件下变性。C)高精度的热力学 将获得单位点BPTI突变体的协同折叠转变 与P.Privalov合作。D)关于BPTI的脉冲交换实验 将进行突变。E)核磁共振和氢交换实验 将在保留14-38的BPT1模拟[14-38]ABU上执行 二硫键，但其他四个半胱氨酸被α-氨基取代 丁酸。我们将对氢交换特性进行表征， [14-18]ABU在pH 4.5-6时的折叠热力学和核磁共振动力学结构 和3-10摄氏度，在这些条件下，它是部分 折叠结构，以及BPTI折叠的早期中间体模型。 计划对[14-38]ABU的变种进行其他核磁共振实验 部分折叠条件，以及pH小于2和5时的[14-38]ABU C度，它形成酸性状态(A状态)的条件。F)核磁共振 两种还原BPTI类似物的结构和氢交换模型 蛋白质的未折叠状态，将被表征。初步 强有力的证据表明，疏水聚集和非随机的，可能 还原BPTI中的非局域核磁共振检测结构。

The long term objective of this grant is to advance fundamental knowledge of the mechanism of protein hydrogen exchange, a unique and versatile probe of protein dynamic structure. Hydrogen exchange in proteins occurs either by internal motions of the folded state, or by global cooperative unfolding, and we call this the two-process model of exchange. To advance understanding of each process, we characterized relationships between hydrogen exchange rates and average (crystal) structure of the folded state, as well as relationships between hydrogen exchange rates and global unfolding. Toward this end, the work funded by this grant since the last competitive renewal produced a significant body of data on dynamics, folding thermodynamics and crystal structure for 8 variants of the small model protein, bovine pancreatic trypsin inhibitor (BPTI). The research plan in this proposal is twofold; i) to bring the data base we have built to bear on further important questions of mechanism of hydrogen exchange in proteins and of the relationship of dynamics measured by hydrogen exchange to protein average structure (crystal or NMR) and to protein folding, and ii) to test our hypothesis relating protein dynamic structure to protein folding, namely, that the native state slow exchange core is the folding core. Specific aims include the following. a) Exchange rates of surface NHs in BPTI mutants will be determined. b) Double mutant combinations of our single-site mutants will be constructed in order to examine BPTI denaturation at low pH. c) Highly accurate thermodynamics for the cooperative folding transition of single site BPTI mutants will be obtained in collaboration with P. Privalov. d) Pulsed exchange experiments on BPTI mutants will be carried out. e) NMR and hydrogen exchange experiments will be carried out on BPT1 analog [14-38]Abu which retains the 14-38 disulfide bond but has the other four cysteines replaced by alpha-amino butyric acid. We will characterize the hydrogen exchange properties, folding thermodynamics and NMR dynamic structure of [14-18]Abu at pH 4.5-6 and 3-10 degree C, conditions under which it is an ensemble of partially folded structures, and a model for early intermediates in BPTI folding. Other NMR experiments are planned for variants of [14-38]Abu under partially folding conditions, and for [14-38]Abu at pH less then 2 and 5 degree C, conditions under which it forms an acid state (A state). f) NMR structures and hydrogen exchange of two analogs of reduced BPTI, models for the unfolded state of the protein, will be characterized. Preliminary evidence strongly suggest hydrophobic clustering and non-random, possibly non-local, NMR-detected structure in reduced BPTI.