Native State Conformational Ensemble of SEM5 SH3 Domain

分子识别是结构生物学中的重要问题之一。这个问题不仅仅是学术上的，因为对识别机制的清晰理解将有助于开发合理的设计策略，以生产临床上重要蛋白质的激动或拮抗变体或配体。虽然很明显，蛋白质的构象波动（或动力学）既影响结合也受结合的影响，但目前还没有定量的预测模型来说明动力学在识别中的作用。作为一个模型系统，本项目研究了秀丽隐杆线虫SEM5 - Sh3结构域的原生阶段构象集合的性质和能量学。我们的具体目标是：1)利用核磁共振检测的氢交换和15N弛豫实验表征其区域稳定性和动力学；2)确定构象波动的协同性，通过测量在Aim 1中表现出差异稳定性的位置上ALA对GLY突变的影响；3)将突变对稳定性的影响与配体结合的影响联系起来，定量描述波动在识别过程中的作用；4)对所选突变体进行结构热力学分析，通过求解突变体的解结构，并根据观测到的NOE约束变化对其柔性区域进行建模，确定不同状态下的能量。这些实验是我们实现长期目标的重要一步，我们的长期目标是建立一个基于结构的模型，用于定量描述分子识别中构象异质性的作用。

Molecular recognition is one of the most important problems in structural biology. This issue is not just academic, as a clear understanding of the mechanism of recognition will facilitate development of rational design strategies to produce agonistic or antagonistic variants or ligand for clinically important proteins. Although it is clear that the conformational fluctuations (or dynamics) of proteins both affect and are affected by binding, no quantitative, predictive model for the role of dynamics in recognition is available. As a model system, this project investigates the nature and energetics of the native stage conformational ensemble of the SEM5 Sh3 domain from C. elegans. Our Specific Aims are: 1) experimental characterization of its region stability and dynamics, using NMR-detected hydrogen exchange and 15N relaxation; 2) determining the cooperativity of the conformational fluctuations, by measuring the effects of ALA to GLY mutations at positions showing differential stability in Aim 1; 3) correlating the mutations' effects on stability with those on ligand binding, to provide a quantitative description for the role of fluctuations in the recognition process; and 4) structural thermodynamic analysis of selected mutants to determine the energy of the different states, by solving the mutants' solution structures and modeling their flexible regions to the observed changes in their NOE constraints. These experiments are an essential step toward our long-term goal, a structure-based model for quantitatively describing the role of conformational heterogeneity in molecular recognition.