SINGLE PROTON EXCHANGE KINETICS IN PROTEINS

这项补助金的长期目标是促进基础知识 蛋白质氢交换的机制，一个独特的和多功能的探针 蛋白质的动态结构。 蛋白质中的氢交换发生在 通过折叠状态的内部运动，或者通过全球合作， 展开，我们称之为交换的双过程模型。 推进 了解每一个过程，我们描述了 氢交换率和折叠的平均（晶体）结构 国家，以及氢交换率和全球 展开 为此，自去年以来，这项赠款资助的工作 竞争性更新产生了大量关于动态的数据， 折叠热力学和晶体结构的8个变种的小 模型蛋白，牛胰蛋白酶抑制剂（BPTI）。 研究 该提案中的计划有两个方面：i）将我们建立的数据库 对氢交换机制的进一步重要问题产生影响， 蛋白质和氢交换动力学的关系 蛋白质平均结构（晶体或NMR）和蛋白质折叠，以及 ii）验证我们关于蛋白质动态结构与蛋白质 折叠，即自然态慢交换核是折叠， 核心 具体目标如下。 （a）地面汇率 将测定BPTI突变体中的NH。 B）以下的双突变体组合： 我们的单位点突变体将被构建，以检查BPTI c）在低pH下的高精度热力学 将获得单位点BPTI突变体的协同折叠转变 与P. Privalov合作。 d）BPTI上的脉冲交换实验 变种人将被执行。 e）NMR和氢交换实验 将在BPT 1类似物[14-38]Abu上进行，Abu保留了14-38 二硫键，但其他四个半胱氨酸被α-氨基取代 丁酸。 我们将描述氢交换性质， [14-18]Abu在pH4.5 -6时的折叠热力学和NMR动力学结构 和3-10摄氏度的条件下，它是一个整体的部分 折叠的结构，并在BPTI折叠的早期中间体的模型。 其他NMR实验计划用于[14-38]Abu的变体， 部分折叠条件，以及[14-38]Abu在pH小于2和5时 C，在此条件下它形成酸状态（A状态）。 f）NMR 还原BPTI的两种类似物的结构和氢交换， 将表征蛋白质的未折叠状态。 初步 有证据强烈表明疏水聚集和非随机，可能 还原BPTI中的非局部NMR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.