Molecular recognition in the streptavidin-biotin system

描述（由申请人提供）：链霉亲和素/生物素复合物涉及生物学中观察到的最强非共价相互作用之一，是研究高亲和力蛋白质/配体相互作用的理想模型系统。链霉亲和素/生物素复合物的高分辨率晶体结构的分析揭示了紧密结合复合物的许多有利的蛋白质-配体接触特征。然而，我们对许多链霉亲和素突变体的广泛的结构和热力学表征强烈表明，直接的蛋白质-配体接触不能完全解释生物素的极紧密结合。我们以前的研究还表明，生物素遵循一个明确的反应坐标在配体结合和解离反应，某些突变可以改变结合/解离反应的活化能势垒，没有任何显着的影响平衡结构。这些结果使我们提出，链霉亲和素平衡动力学，或结构波动，有助于确定平衡结合能和活化能垒。我们将通过对野生型和选定的突变链霉亲和素/生物素复合物的各向异性温度因子进行分子动力学模拟和详细的晶体学分析来测试这一假设，然后寻找野生型与突变复合物的结构波动和量热测量之间的相关性。我们以前的研究结果还表明，特定的水分子在生物素结合/解离反应中起着关键的能量作用。我们将使用分子动力学模拟来测试这一建议，进一步表征水在链霉亲和素结合位点的相互作用。我们将使用这些模拟结果，建议突变，可以改变平衡水的行为在复杂的，从而调节配体结合自由能和/或活化能。这些研究将增强我们对高亲和力蛋白质/配体结合相互作用的理解，并将帮助我们阐明在基于结构的配体设计项目中应该有用的重要概念。这些信息将有助于合理的药物设计应用于治疗上重要的蛋白质靶点。

DESCRIPTION (provided by applicant): The streptavidin/biotin complex involves one of the strongest non-covalent interactions observed in biology, and is an ideal model system for the study of high-affinity protein/ligand interactions. Analysis of high- resolution crystal structures for the streptavidin/biotin complex reveals numerous favorable protein-ligand contacts characteristic of a tightly bound complex. However, our extensive structural and thermodynamic characterization of numerous streptavidin mutants strongly suggests that direct protein-ligand contacts cannot fully explain the extremely tight biotin binding. Our previous studies also suggest that biotin follows a well defined reaction coordinate during ligand binding and dissociation reactions, and that certain mutations can alter the activation energy barrier for the binding/dissociation reactions, without any significant effect on the equilibrium structure. These results lead us to propose that streptavidin equilibrium dynamics, or structural fluctuations, help determine the equilibrium binding energy and activation energy barrier. We will test this hypothesis by performing molecular dynamics simulations and detailed crystallographic analyses of anisotropic temperature factors for wild-type and selected mutant streptavidin/biotin complexes, and then look for correlations between structural fluctuations and calorimetric measurements in wild-type versus mutant complexes. Our previous results also suggest that specific water molecules play a key energetic role in the biotin binding/dissociation reactions. We will use molecular dynamics simulations to test this proposal by further characterizing water interactions in the streptavidin binding site. We will use these simulation results to suggest mutations that can alter the equilibrium water behavior in the complex, and thus modulate the ligand binding free energy and/or activation energy. These studies will enhance our understanding of high-affinity protein/ligand binding interactions, and will help us elucidate important concepts that should be useful in structure-based ligand design projects. Such information will be helpful in rational drug design applications for therapeutically important protein targets.