Protein-Ligand Binding Equilibrium and Mechanism under Macromolecular Crowding

描述(申请人提供)：分子识别是生物系统中的一个基本过程，为外部输入，如小分子配体，提供了一种转化为细胞反应的手段。许多药物通过模仿配体并与目标蛋白质结合来利用这一现象。在配体结合的过程中，蛋白质往往会发生构象变化。支撑这一转变的机制可以被认为是两条主要途径的组合，称为“诱导匹配”和“构象采样”。确定这些途径的贡献可能很困难，因为这些途径沿途的中间状态存在于大多数技术无法检测到的种群中。此外，目前尚不清楚拥挤的环境，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。我将使用几种技术来研究构象选择对两种周质结合蛋白的配基结合亲和力的贡献。我将通过顺磁松弛增强来量化稀疏和拥挤条件下两种不同非结合蛋白状态的数量。预计这些结果将与一种基于模拟的方法相结合，使用我们实验室开发的一种称为“后处理”的技术来预测由于拥挤而导致的构象之间平衡的预期变化。配基滴定将在类似的条件下进行，由内在色氨酸荧光监测，以评估结合亲和力。这些结果将得到模拟的补充，这些模拟旨在为连接和非连接形式的周质结合蛋白的关闭和打开过程产生平均作用力的势。这个模拟的结果将被分析以确定蛋白质的开放形式和闭合形式的结合亲和力的比率，并将在稀疏和拥挤的条件下重复以预测总亲和力的变化。该项目将产生一些第一批研究，将非结合状态下直接观察到的构象平衡与配体结合性质联系起来。在溶液中使用聚集剂将使我们深入了解稀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。 公共卫生相关性：研究分子识别对于理解信号过程如何在生命系统中发挥作用至关重要。增加对配体结合的知识将导致对蛋白质-配体相互作用不能正常发挥作用时的更多洞察力，这通常会导致导致疾病状态的不成功信号通路。周质结合蛋白为研究类细胞条件下的构象变化和配体结合提供了一个很好的模型系统，其结果将创建一个更全面的生命系统中分子识别过程的图景。

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. PUBLIC HEALTH RELEVANCE: Study of molecular recognition is essential to understanding how signaling processes function in living systems. Increased knowledge of ligand binding will result in additional insight when protein-ligand interactions do not function properly, often leading to unsuccessful signaling pathways that can cause disease states. Periplasmic binding proteins provide an excellent model system for investigating conformational changes and ligand binding in cell-like conditions, the results of which will create a more comprehensive picture of molecular recognition processes in living systems.