BIOPHYSICAL STUDIES OF FOLDING MUTANTS OF STAPH NUCLEASE

描述（由申请人提供）：本项目的主要目的是定量描述驱动葡萄球菌核酸酶氨基酸序列进入其三维结构的物理化学。 当核酸酶不折叠时持续存在的结构的实验研究将采用NMR光谱来测量反映在残余偶极偶联（RDC）中的长程结构特征。 先前的RDC数据已经证明，天然样拓扑结构”在变性核酸酶中持续存在，即使在10个大的疏水残基突变后存在8 M尿素。 虽然这一结论的论据是令人信服的，但需要对这些耦合中的信息进行更多的定量理解，以完成对许多相互转换构象的这种知之甚少的集合的描述。 两种数据解释的策略，将追求不依赖于单一结构的总体平均结构的表示。 为了获得尽可能详细的结构，将收集具有不同排列张量的许多组RDC，使用电场或化学修饰来改变排列张量。 尝试将在葡萄球菌核酸酶和其他三种蛋白质（泛素，eglin C，和fyn-SH 3结构域），以证明一个天然样的拓扑结构在紧凑的变性状态的偶极耦合从天然和变性状态的直接相关。 一种新的策略，预测新的蛋白质折叠的结构，基于建模侧链/骨架相互作用与phi/psi/chil倾向，将追求。 CASP 5的初步成功表明，更好地采样螺旋和链之间的转弯构象可能会导致在低分辨率下预测新折叠的重大进展。 最近开发的统计潜力的phi/psi/chil角和局部侧链/侧链的相互作用将结合扭转角动力学和应用于蛋白质结构的预测在更高的分辨率，在细化的从头模型和同源模型。

DESCRIPTION (provided by applicant): The principal objective of this project is a quantitative description of the physical chemistry that drives the amino acid sequence of staphylococcal nuclease into its three dimensional structure. Experimental studies of structure that persists when nuclease is not folded will employ NMR spectroscopy to measure long range structural features reflected in residual dipolar couplings (RDCs). Previous RDC data have demonstrated that a native-like topology" persists in denatured nuclease, even in the presence of 8 M urea after mutation of 10 large hydrophobic residues. While the argument is compelling for this conclusion, a much more quantitative understanding of the information in these couplings is needed to complete the picture of this poorly understood ensemble of many inter-converting conformations. Two strategies of data interpretation will be pursued that do not rely on single structures for representation of the ensemble average structure. To achieve the most detailed structure possible, many sets of RDCs will be collected with different alignment tensors, using electric fields or chemical modification to alter the alignment tensor. Attempts will be made in staphylococcal nuclease and three other proteins (ubiquitin, eglin C, and fyn-SH3 domain) to demonstrate a native-like topology in compact denatured states by direct correlation of dipolar couplings from the native and the denatured states. A novel strategy for predicting the structure of new protein folds, based on modeling side-chain/backbone interactions with phi/psi/chil propensities, will be pursued. Initial successes at CASP5 suggest that better sampling of the conformations of turns between helices and strands could lead to significant advances in predicting new folds at low resolution. Recently developed statistical potentials for phi/psi/chil angles and for local side-chain/side-chain interactions will be combined with torsion angle dynamics and applied to the prediction of protein structures at higher resolution, in refinement of both de novo models and homology models.