BIOPHYSICAL STUDIES OF FOLDING MUTANTS OF STAPH NUCLEASE

描述(申请人提供)：这个项目的主要目标是对驱动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。对核酸酶没有折叠时持续存在的结构的实验研究将使用核磁共振光谱来测量反映在剩余偶极耦合(RDC)中的远程结构特征。以前的RDC数据已经证明，在变性核酸酶中，即使在10个大的疏水残基突变后存在8M尿素的情况下，也仍然存在天然的类拓扑结构。虽然这一结论的论点很有说服力，但需要对这些偶联中的信息有更多的定量理解，才能完整地描绘出这一鲜为人知的许多相互转化构象的集合。将采用两种不依赖单一结构来表示集合平均结构的数据解释战略。为了获得尽可能详细的结构，将收集具有不同排列张量的多组RDC，使用电场或化学修饰来改变排列张量。我们将试图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)中通过直接关联来自天然状态和变性状态的偶极偶极偶联来展示紧凑变性状态下的天然类拓扑结构。将寻求一种新的策略来预测新的蛋白质折叠的结构，该策略基于对具有phi/psi/chil倾向的侧链/主干相互作用的建模。在CASP5上取得的初步成功表明，对螺旋和链之间的旋转构象进行更好的采样可以在预测低分辨率新折叠方面取得重大进展。最近发展的phi/psi/chil角和局部侧链/侧链相互作用的统计势将与扭角动力学相结合，并在改进从头模型和同源模型的基础上，应用于更高分辨率的蛋白质结构预测。

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.