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: 6627295
• 负责人: DAVID Robert SHORTLE
• 金额: $ 36.56万
• 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/6627295
• 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.

该项目的主要目标是定量描述将葡萄球菌核酸酶的氨基酸序列与其三维结构连接起来的物理化学。 所采用的两种方法是部分折叠构象的结构和动力学的核磁共振表征以及估计其热力学性质的计算机模拟。先前对变性状态片段模型的研究令人惊讶地发现，它表现出与折叠核酸酶相同的拓扑或低分辨率结构。 为了更详细地了解维持这种高度动态结构的相互作用，将利用用氘取代所有碳结合氢而使检测 HN-HN NOES 的灵敏度提高 15 至 50 倍。 此外，还将在部分取向的样品上测量残余偶极耦合。 核酸酶的初始平衡折叠途径将使用基于 TROSY-HSQC 的 NMR 实验扩展到更高分辨率，以跟踪肽链的自组织作为甘油浓度的函数。 负责该组织的特定链-链相互作用将直接通过 NOES 或其他结构参数来识别，或者通过对序列修改产生的结构/动力学敏感的 NMR 参数的相关变化来间接识别。 最近对四种核酸酶突变体中氢交换的研究已经确定了熔球状态在 m 值效应（对变性剂敏感性的变化）中的作用。 通过氢交换、NMR 参数和荧光对其他 m+ 和 m- 突变体进行分析，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，即天然状态的拓扑部分是由二级结构片段堆积的高熵决定的，蒙特卡罗采样方法被用来估计接近真实天然结构和接近严重错误折叠结构的低能量构象的密度。 对于几种小螺旋蛋白，两种独立的模拟策略证明了野生拓扑结构的更高密度的构象。 未来的工作将完善计算机模型，解决含有β链的蛋白质，并开发和测试一种策略，通过从头构建具有最大片段包装熵的折叠，从序列加二级结构预测蛋白质的低分辨率结构。

项目成果

Genetic analysis of staphylococcal nuclease: identification of three intragenic "global" suppressors of nuclease-minus mutations.

葡萄球菌核酸酶的遗传分析：鉴定核酸酶缺失突变的三个基因内“全局”抑制因子。

• DOI: 10.1093/genetics/110.4.539
• 发表时间: 1985
• 期刊: Genetics
• 影响因子: 3.3
• 作者: Shortle,D;Lin,B
• 通讯作者: Lin,B

Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene.

酵母肌动蛋白基因温度敏感突变等位基因的构建和遗传特征。

• DOI: 10.1073/pnas.81.15.4889
• 发表时间: 1984
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Shortle,D;Novick,P;Botstein,D
• 通讯作者: Botstein,D