Native State Conformational Ensemble of SEM5 SH3 Domain

分子识别是结构生物学中最重要的问题之一。这个问题不仅仅是学术上的问题，因为对识别机制的清楚理解将有助于开发合理的设计策略来为临床上重要的蛋白质产生激动型或拮抗型变体或配体。虽然蛋白质的构象波动(或动力学)显然既影响结合又受结合的影响，但还没有关于动力学在识别中的作用的定量预测模型。作为一个模型系统，本项目研究了线虫SEM5 SH3结构域的自然阶段构象系综的性质和能量学。我们的具体目标是：1)利用核磁共振检测到的氢交换和15N驰豫，对其区域稳定性和动力学进行实验表征；2)通过测量Aim 1中显示差异稳定性的位置上Ala对Gly突变的影响，确定构象波动的协同性；3)将突变对稳定性的影响与对配体结合的影响相关联，为波动在识别过程中的作用提供定量描述；以及4)对选定的突变体进行结构热力学分析，通过求解突变体的溶液结构并根据观察到的NOE约束的变化来模拟它们的柔性区域，以确定不同状态的能量。这些实验是朝着我们的长期目标迈出的重要一步，我们的长期目标是一个基于结构的模型，用于定量描述构象异质性在分子识别中的作用。

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.