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性的，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，以产生临床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（或动态）的影响，并受结合，没有定量的，预测模型的动态识别中的作用是可用的。 作为一个模型系统，本项目研究了C.优雅 我们的具体目标是：1）使用NMR检测的氢交换和15 N弛豫对其区域稳定性和动力学的实验表征; 2）通过测量在Aim 1中显示差异稳定性的位置处ALA至GLY突变的影响来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，对波动在识别过程中的作用进行定量描述;和4）所选突变体的结构热力学分析以确定不同状态的能量，通过求解突变体的解结构，并根据NOE约束的观察变化对其柔性区域进行建模。 这些实验是朝着我们的长期目标迈出的重要一步，这一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.