Collaborative Research: Small molecules as chemical probes of protein dynamics and protein-protein interactions

了解活细胞如何发出信号和协调复杂的细胞活动的细节是一个尚未解决的具有高度基础意义的问题。众所周知，细胞是由一系列分子组成的，因此这些分子在特定时间和位置的相互作用可能对功能结果负责。在原子尺度上量化一对分子之间的分子间相互作用仍然具有挑战性，这主要是由于对潜在动态运动的理解不足。重要的是，生物分子（如蛋白质）中的许多结构运动不能直接从高分辨率静态实验结构中推断出来，因此需要新的方法来探测动态运动。在这个项目中，原子分辨分子模拟与各种实验技术的独特结合建立了研究蛋白质-蛋白质相互作用的新方法，并且开发的小分子可能导致控制这种相互作用的工具。从事该项目的研究生和博士后研究人员在蛋白质的计算机模拟方法和涉及蛋白质表征和光谱方法的实验方法方面接受跨学科培训，从而了解理论和实验可以相互告知的方式。该项目的外展活动通过动手实验室体验来教育和培训STEM学科的高中生和教师。由此产生的工具，如软件、结构模型和蛋白质结构，在化学工程、生物物理学和药理学课程中得到展示，并通过研讨会、专题讨论会和会议在更广泛的科学界中传播。本研究项目采用蛋白质全原子模型的大规模分子模拟技术来推断潜在的动力学，发现隐藏的构象状态，并量化蛋白质-蛋白质界面的相互作用。各种各样的计算方法被追求，包括在显式溶剂中的经典分子动力学模拟，增强构象采样和蛋白质热力学表征的方法，以及小分子对接的蒙特卡罗协议。为了更好地理解动力学和相互作用，建模和模拟工作与许多实验（生化、生物物理和光谱）技术在不同层次上相结合。这些组合工具用于研究g蛋白信号传导（RGS）蛋白的调节因子，使用噻二唑烷酮（TDZD）类似物作为小分子化学探针。重点是量化三种不同RGS蛋白的动力学差异，从而导致不同小分子的特异性和效力差异，并发现小分子影响和抑制各种RGS蛋白与g蛋白活化的g - α亚基之间相互作用的机制。该蛋白家族的机制和相互作用的量化可能揭示动态运动在调节蛋白质-蛋白质相互作用中的作用程度，以及这种运动可以用于靶向蛋白质-蛋白质界面的独特方式。

Understanding details of how living cells signal and coordinate complex cellular activities is an unsolved problem of high fundamental significance. It is known that cells are comprised of an array of molecules, and therefore the interactions among these molecules at a specific time and location are potentially responsible for functional outcomes. It remains challenging to quantify intermolecular interactions between a pair of molecules at atomic scale, owing largely to a poor understanding of underlying dynamic motions. Importantly, many such structural motions in biomolecules such as proteins cannot be directly inferred from high-resolution static experimental structures, and therefore novel methods to probe dynamic motions are needed. In this project, the unique combination of atomically-resolved molecular simulations with various experimental techniques establishes new approaches to study protein-protein interactions, and the small molecules developed may lead to tools for controlling such interactions. The graduate and postdoctoral researchers working on this project acquire interdisciplinary training in computer simulation approaches for proteins and experimental approaches involving protein characterization and spectroscopic methods, thereby gaining an appreciation for ways in which theory and experiment can inform each other. The outreach activities of this project educate and train high school students and teachers in STEM disciplines via hands-on lab experiences. The resulting tools such as software, structural models, and protein constructs are demonstrated in chemical engineering, biophysics, and pharmacology courses, and are disseminated among the broader scientific community via workshops, symposia, and meetings.This research project employs large-scale molecular simulation techniques on all-atom models of proteins to infer underlying dynamics, discover hidden conformational states, and quantify interactions at protein-protein interfaces. A variety of computational approaches are pursued including classical molecular dynamics simulations in explicit solvent, methods for enhanced conformational sampling and thermodynamic characterization of proteins, and Monte Carlo protocols for small molecule docking. To gain a better understanding of dynamics and interactions, the modeling and simulation effort are integrated with many experimental (biochemical, biophysical, and spectroscopic) techniques at various levels. These combined tools are used to study regulators of G-protein signaling (RGS) proteins using thiadiazolidinone (TDZD) analogues as small molecule chemical probes. The focus is to quantify differences in dynamics of three-different RGS proteins that result in differences in specificity and potency for different small molecules, and discover the mechanisms by which small molecules affect and inhibit interactions between various RGS proteins and activated G-alpha subunits of G-proteins. The quantification of mechanisms and interactions in this family of proteins may reveal the extent to which dynamic motions play a role in regulating protein-protein interactions, and the unique ways in which such motions can be exploited for targeting protein-protein interfaces.