BIOPHYSICAL STUDIES OF FOLDING MUTANTS OF STAPH NUCLEASE

葡萄球菌核酸酶折叠突变体的生物物理学研究

• 批准号: 6050993
• 负责人: DAVID Robert SHORTLE
• 金额: $ 37.55万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-05-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6050993
• 关键词: Staphylococcus; biophysics; chemical kinetics; computer simulation; conformation; deuterium; dipole moment; enzyme structure; fluorescence spectrometry; hydropathy; mutant; nuclear magnetic resonance spectroscopy; nuclease; point mutation; protein denaturation; protein folding; protein sequence; structural biology; thermodynamics

The principal objective of this project is a quantitative description of the physical chemistry that connects the amino acid sequence of staphylococcal nuclease to its three dimensional structure. The two approaches taken are NMR characterization of the structure and dynamics of partially folded conformations and computer simulation to estimate their thermodynamic properties. Previous studies of a fragment model of the denatured state have revealed, quite surprisingly, that it exhibits the same topology or low resolution structure as folded nuclease. To obtain a more detailed picture of the interactions that maintain this highly dynamic structure, advantage will be taken of the 15 to 50 fold increase in sensitivity for detecting HN-HN NOES that results from replacing all carbon-bound hydrogens with deuterium. In addition, residual dipolar couplings will be measured on partially oriented samples. An initial equilibrium folding pathway of nuclease will be extended to higher resolution using NMR experiments based on the TROSY-HSQC to follow the self organization of the peptide chain as a function of glycerol concentration. The specific chain-chain interactions responsible for this organization will be identified either directly through NOES or other structural parameters, or indirectly through correlated changes in NMR parameters sensitive to structure/dynamics produced by modifications in sequence. Recent studies of hydrogen exchange in four nuclease mutants have identified a role for the molten globule state in m-value effects --changes in sensitivity to denaturants. Analysis of additional m+ and m- mutants by hydrogen exchange, NMR parameters, and fluorescence will establish a quantitative relationship between m-values and changes in population/structure of this molten folding intermediate. To test this the hypothesis that the topology of the native state is determined in part by a high entropy of packing of secondary structural segments, Monte Carlo sampling methods are being used to estimate the density of low energy conformations near the true native structure and near grossly misfolded structures. For several small helical proteins, two independent simulation strategies demonstrate a higher density of conformations with the wild-topology. Future work will refine the computer model, address beta-strand containing proteins, and develop and test a strategy for predicting the low resolution structure of proteins from sequence plus secondary structure, through de novo construction of folds with maximal segment-packing entropy.

本项目的主要目标是定量描述葡萄球菌核酸酶的氨基酸序列与其三维结构之间的物理化学关系。 所采取的两种方法是NMR表征的结构和动力学的部分折叠构象和计算机模拟，以估计其热力学性质。以前的研究表明，片段模型的变性状态，相当令人惊讶的是，它表现出相同的拓扑结构或低分辨率的结构作为折叠的核酸酶。 为了获得维持这种高度动态结构的相互作用的更详细的图片，将利用由用氘取代所有碳结合的氢导致的检测HN-HN NOES的灵敏度增加15至50倍的优势。 此外，将在部分定向样品上测量残余偶极耦合。 使用基于TROSY-HSQC的NMR实验，将核酸酶的初始平衡折叠途径扩展到更高的分辨率，以遵循作为甘油浓度的函数的肽链的自组织。 负责该组织的特定链-链相互作用将通过NOES或其他结构参数直接鉴定，或通过对序列中的修饰产生的结构/动力学敏感的NMR参数的相关变化间接鉴定。 最近在四种核酸酶突变体中的氢交换研究已经确定了熔融球状态在m值效应中的作用-对变性剂敏感性的变化。 通过氢交换、NMR参数和荧光分析额外的m+和m-突变体将建立m值与该熔融折叠中间体的群体/结构变化之间的定量关系。 为了检验这一假设，即天然状态的拓扑结构部分地由二级结构片段的堆积的高熵决定，蒙特卡罗采样方法被用于估计真实天然结构附近和严重错误折叠结构附近的低能量构象的密度。 对于几个小的螺旋蛋白，两个独立的模拟策略表现出更高的密度与野生拓扑结构的构象。 未来的工作将完善的计算机模型，地址β-链包含蛋白质，并开发和测试的战略预测低分辨率结构的蛋白质的序列和二级结构，通过从头构建折叠最大片段包装熵。