BIOPHYSICAL STUDIES OF FOLDING MUTANTS OF STAPH NUCLEASE

描述（由申请人提供）：该项目的主要目标是对葡萄球菌核酸酶的氨基酸序列进入其三维结构的物理化学进行定量描述。对核酸酶未折叠时结构的实验研究将采用核磁共振波谱法来测量残留偶极偶联（rdc）中反映的远程结构特征。先前的RDC数据表明，即使在10个大型疏水残基突变后存在8 M尿素的情况下，变性核酸酶中仍存在类似天然结构的拓扑结构。虽然这个结论的论点是令人信服的，但需要对这些耦合中的信息进行更定量的理解，以完成对许多相互转换的构象的知之甚少的集合的图像。将采用两种数据解释策略，它们不依赖于单个结构来表示总体平均结构。为了获得尽可能详细的结构，将收集许多组具有不同取向张量的rdc，使用电场或化学修饰来改变取向张量。将尝试在葡萄球菌核酸酶和其他三种蛋白质（泛素、eglin C和fyn-SH3结构域）中，通过天然状态和变性状态的偶极偶联的直接相关，来证明紧凑变性状态下的类似天然状态的拓扑结构。基于侧链/主链相互作用与phi/psi/ child倾向的建模，将寻求一种预测新蛋白质折叠结构的新策略。CASP5的初步成功表明，对螺旋和链之间的旋转构象进行更好的采样，可能会在低分辨率下预测新的褶皱方面取得重大进展。最近开发的phi/psi/chil角和局部侧链/侧链相互作用的统计势将与扭转角动力学相结合，并应用于更高分辨率的蛋白质结构预测，以改进新模型和同源模型。

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.