Protein-Ligand Binding Equilibrium and Mechanism under Macromolecular Crowding

描述（由申请人提供）：分子识别是生物系统中的一个重要过程，为将外部输入（如小分子配体）转化为细胞反应提供了一种手段。许多药物通过模拟配体并结合靶蛋白来利用这一现象。在与配体结合的过程中，蛋白质经常发生构象变化。支持这种转变的机制可以被认为是两个主要途径的组合，称为“诱导拟合”和“构象采样”。“确定这些途径的贡献可能很困难，因为大多数技术无法检测到这些途径的中间状态沿着存在于种群中。此外，目前还不知道拥挤的环境，如细胞内部，将如何影响这些中间状态或蛋白质对其配体的亲和力。我将使用分子生物学技术来研究构象选择对两种周质结合蛋白的配体结合亲和力的贡献。我将量化人口的两个不同的未结合的蛋白质状态在稀释和拥挤的条件下，顺磁弛豫增强。预计这些结果将与基于模拟的方法相结合，使用我们实验室开发的称为“后处理”的技术来预测拥挤导致的构象之间的预期平衡变化。将在相似条件下进行配体滴定，通过固有色氨酸荧光监测以评估结合亲和力。这些结果将补充模拟设计，以产生一个潜在的平均力的封闭和开放过程中的配体和unliganded形式的周质结合蛋白。将分析该模拟的结果以确定蛋白质的开放和封闭形式的结合亲和力的比率，并将在稀释和拥挤条件下重复以预测总体亲和力的变化。这个项目将产生一些第一个研究，直接观察到的构象平衡在未结合状态的配体结合特性。在溶液中使用拥挤剂将深入了解稀释溶液研究和生物条件之间的差异，并加强我们对生物系统中发生的分子识别的理解。

DESCRIPTION (provided by applicant): Molecular recognition is an essential process in biological systems, providing a means for external inputs, such as small molecule ligands, to be translated into cellular responses. Many drugs take advantage of this phenomenon by mimicking ligands and binding to target proteins. During the process of ligand binding, proteins often undergo conformational changes. The mechanism underpinning this transition can be considered as a combination of two major pathways, known as "induced fit" and "conformational sampling." Determining contribution from these routes can be difficult, as intermediate states along the pathways exist at populations that most techniques cannot detect. Additionally, it is not known how a crowded environment, such as the inside of a cell, will affect these intermediate states or the affinity of proteins for their ligands as a result. I will be using seveal techniques to investigate the contribution of conformational selection to ligand binding affinity fr two periplasmic binding proteins. I will quantify the populations of two distinct unbound protein states in both dilute and crowded conditions by paramagnetic relaxation enhancement. It is expected that the These results will be combined with a simulation-based approach, using a technique developed in our lab known as "postprocessing" to predict the expected shift in equilibrium between conformations as a result of crowding. Ligand titrations will be performed in similar conditions, monitored by intrinsic tryptophan fluorescence to assess binding affinities. These results will be complemented with simulations designed to generate a potential of mean force for the closing and opening process of periplasmic binding proteins in liganded and unliganded forms. The results of this simulation will be analyzed to determine the ratios of binding affinities of open and closed forms of the proteins, and will be repeated in dilute and crowded conditions to predict changes in overall affinity. This project will produce some of the first studies that connect directly observed conformational equilibria in the unbound state to ligand binding properties. Using crowding agents in solution will provide insight into differences between dilute solution studies and biological conditions, and strengthen our understanding of molecular recognition as it occurs in biological systems.