BIOPHYSICAL STUDIES OF FOLDING MUTANTS OF STAPH NUCLEASE

该项目的主要目标是对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进行定量描述。采用的两种方法是核磁共振表征部分折叠构象的结构和动力学和计算机模拟来估计它们的热力学性质。先前对变性状态片段模型的研究显示，相当令人惊讶的是，它表现出与折叠核酸酶相同的拓扑结构或低分辨率结构。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像，将利用探测HN-HN NOES的灵敏度提高15到50倍的优势，这种灵敏度是用氘取代所有碳结合氢的结果。此外，残余偶极耦合将测量部分取向样品。利用基于TROSY-HSQC的核磁共振实验，将核酸酶的初始平衡折叠途径扩展到更高分辨率，以跟踪肽链的自组织作为甘油浓度的函数。造成这种组织的特定链-链相互作用可以直接通过NOES或其他结构参数来识别，也可以通过对序列修改产生的结构/动力学敏感的核磁共振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、核磁共振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人口/结构变化之间的定量关系。为了验证这一假设，即天然状态的拓扑结构部分由二级结构段的高熵填充决定，蒙特卡罗采样方法被用于估计真正天然结构附近和严重错误折叠结构附近的低能构象的密度。对于一些小螺旋蛋白，两种独立的模拟策略显示出具有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，定位含有β链的蛋白质，并开发和测试一种策略，通过重新构建具有最大片段填充熵的折叠，从序列加二级结构预测蛋白质的低分辨率结构。

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.