Native State Conformational Ensemble of SEM5 SH3 Domain

SEM5 SH3 结构域的天然态构象集合

• 批准号: 6361146
• 负责人: VINCENT J. HILSER
• 金额: $ 22.47万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6361146
• 关键词: calorimetry; chemical stability; chemical synthesis; computer simulation; fluorescence spectrometry; high performance liquid chromatography; intermolecular interaction; model design /development; molecular dynamics; molecular shape; molecular site; nuclear magnetic resonance spectroscopy; physical model; polymerase chain reaction; protein purification; protein structure function; proteins; protonation; site directed mutagenesis; structural biology; surface property; thermodynamics

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.

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问题，因为对识别机制的清晰理解将有助于制定合理的设计策略，以产生临床重要蛋白质的激动或拮抗变体或配体。 尽管很明显蛋白质的构象波动（或动力学）既影响结合又受结合影响，但对于动力学在识别中的作用还没有定量的预测模型。 作为一个模型系统，该项目研究了秀丽隐杆线虫 SEM5 Sh3 结构域的原生阶段构象系综的性质和能量学。 我们的具体目标是：1) 使用 NMR 检测的氢交换和 15N 弛豫对其区域稳定性和动力学进行实验表征； 2) 通过测量 ALA 对 GLY 突变在目标 1 中显示差异稳定性的位置的影响，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，为识别过程中波动的作用提供定量描述； 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察到的 NOE 约束变化对其柔性区域进行建模，来确定不同状态的能量。 这些实验是实现我们长期目标的重要一步，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。