NMR STUDIES OF PROTEIN FLUCTUATION AND FOLDING

蛋白质波动和折叠的核磁共振研究

基本信息

批准号：
3280278
负责人：
NEVILLE Robert KALLENBACH
金额：
$ 8.62万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-04-01 至 1988-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3280278
关键词：
chemical reaction conformation disulfide bond hydrogen transport insect poison ligands myoglobin neurotoxins nuclear magnetic resonance spectroscopy organic base peptides solutions

项目摘要

1H NMR investigations of the nature of fluctuations in structure of native protein molecules responsible for the chemical exchange of N-H protons with solvent are proposed. Two model systems will be used. First I have prepared a set of closely related short peptides (18-24 amino acids) from lyophilized venom of the honey bee, A. mellifera. These include the central nervous neurotoxin apamin, the mast cell degranulating peptide (P401), and two molecules of unknown function, secapin and tertiapin (1-3). Each represents a minor component (less than 3 percent) of the total venom compared to mellitin (greater than 50 percent) and phospholipase A. Their structure, stability and spectroscopic properties (4-6) make them ideal models for Alpha-helices in solution; in each case the stability is very high due to the presence of disulfide bridges. Measurements of 1H-2H exchange rates as functions of concentration, pH and temperature using the fully assigned amide IH NMR spectrum of apamin (7) should provide direct tests of the magnitude, cooperativity, and possibly kinetics of the "opening" reactions mediating hydrogen exchange in these molecules. Studies of the folding of these chains can also be carried out, since apamin, and possibly the others, refold following reduction and reoxidation of the disulfides (4,7). The second experimental system involves the use of general base catalysts to catalyse the exchange of certain side chain residues in myoglobin and other heme proteins. In a number of heme proteins, the 1H resonances corresponding to exchangeable side chains and peptides in the vicinity of the heme have been assigned (10). These include several histidine ring N-H protons, which I have shown to respond to catalysis by organic bases (e.g., piperidine) as well as hydroxide. Preliminary results indicate that the simple local unfolding model fails to explain the differences in catalysis observed for piperidine and OH-. Further work using other basis and experimental conditions, together with a model for the "open" state of the ligand -- the aquo cobalamin imidazole complex -- should provide the first detailed picture of the exchange reaction in which access to the extremely hydrophobic heme pocket is involved.

1H NMR对天然结构波动的性质的研究负责N-H质子化学交换的蛋白质分子与提出了溶剂。将使用两个模型系统。首先我有从中准备了一组密切相关的短肽（18-24个氨基酸）蜂蜜蜜蜂的冻干毒液，A。Mellifera。这些包括中枢神经神经毒素apamin，肥大细胞脱粒肽（p401）和两个未知功能的分子，secapin和tertiapin （1-3）。每个代表次要组成部分（少于3％）与梅林蛋白（大于50％）相比，毒液的总毒液和磷脂酶A.它们的结构，稳定性和光谱特性（4-6）使其成为溶液中α-螺旋的理想模型；在每种情况下由于存在二硫键，稳定性非常高。 1H-2H汇率的测量作为浓度，pH和pH和使用完全指定的酰胺IH NMR光谱的温度（7）应直接测试大小，合作性，甚至可能 “开放”反应的动力学介导了这些反应分子。这些链的折叠的研究也可以进行，由于apamin，甚至其他可能是Apamin，因此在减少后重新折叠二硫化物的重新氧化（4,7）。第二个实验系统涉及使用一般碱催化剂来催化交换肌红蛋白和其他血红素蛋白中的某些侧链残基。在血红素蛋白的数量，与可交换的1H共振血红素附近的侧链和肽已分配（10）。这些包括几个组氨酸环n-h质子，我已经显示响应有机碱（例如哌啶）以及氢氧化物。初步结果表明简单的本地展开模型无法解释哌啶观察到的催化差异哦 - 。使用其他基础和实验条件进一步工作，以及配体“开放”状态的模型 - aquo 钴胺素咪唑综合体 - 应提供第一张详细图片进入极端疏水性血红素的交换反应涉及口袋。