Collaborative Research: Systematic Investigation of the Structure, Dynamics, and Energetics of Hydrogen Bonds and the Protein Interior Using Ketosteroid Isomerase and Model Systems

蛋白质通过极大地加速特定的化学反应（催化），与蛋白质和其他配体结合以及通过其他机制发挥作用;这些作用对所有生命过程都是必不可少的。 用于酶催化、蛋白质调节和蛋白质相互作用的物理机制是了解蛋白质功能的基础。在最深层次上解决这些物理机制，需要了解基本的力量和蛋白质内结构，动力学和能量学的相互作用。因此，这种理解将产生广泛而深刻的影响。由于理解这些潜在的力量和由此产生的大分子行为具有巨大的挑战性，需要多学科，高度集成的方法，该项目涉及一个多学科团队，具有广泛而深入的专业知识，允许使用，集成和创新的方法，包括结构-功能和能量探针。这一提议将有助于培养一批具有科学严谨性和完整性的多样化和高度影响力的年轻科学家。 该项目还将在研究生研究型大学和代表性不足的少数民族丰富的本科院校之间实施一种新颖的、具有高度影响力的研究整合，协同增强研究和指导。该研究重点是酶酮甾体异构酶（KSI）作为一种异常强大的系统，提出和剖析蛋白质功能和能量学中的基本问题，并将使用KSI和其他蛋白质和模型系统来理解氢键结构和能量学，并确定整个蛋白质支架的结构，动力学和能量学的相互作用。氢键可能是生物学中最基本的相互作用，但我们对氢键和氢键网络的结构、能量学和动力学的理解和预测能力非常有限。在这项研究中，氢键结构的定量和预测模型将被开发，重点是决定氢键长度和耦合的因素;蛋白质环境中的氢键能量学。 利用的广泛的方法包括定点诱变和非天然氨基酸掺入;通过NMR和低温和室温X射线以及中子晶体学的蛋白质结构和动力学;通过量子力学/分子力学（QM/MM）的原子级能量学、动力学和催化;并通过1H NMR、等温滴定量热法、小分子中子和X射线衍射评估小分子氢键（H键）能量学和配体缔合，和量子力学。该项目得到了生物科学理事会分子和细胞生物科学司分子生物物理学小组的支持。

Proteins function by greatly accelerating specific chemical reactions (catalysis), binding to proteins and other ligands, as well as via other mechanisms; these actions are essential for all life processes. The physical mechanisms used for enzyme catalysis, protein regulation, and protein interactions, are fundamental to understanding protein function. Addressing these physical mechanisms at the deepest level, requires understanding basic forces and the interplay of structure, dynamics, and energetics within proteins. Thus, such understanding will have widespread and profound consequences. As understanding these underlying forces and the resulting macromolecular behaviors has been enormously challenging and will require multidisciplinary, highly integrated approaches, this project involves a multidisciplinary team with broad and deep expertise that allows the use, integration, and innovation of approaches encompassing structure-function and energetic probes. This proposal will aid in training of a diverse and highly impactful group of young scientists with scientific rigor and integrity. This project will also implement a novel, highly impactful integration of research between graduate research universities and underrepresented minority-rich undergraduate institutions, synergistically enhancing research and mentoring.This research focuses on the enzyme ketosteroid isomerase (KSI) as an exceptionally powerful system to pose and dissect fundamental questions in protein function and energetics, and will use KSI and other protein and model systems to understand hydrogen bond structure and energetics and to determine the interplay of structure, dynamics, and energetics throughout a protein scaffold. The hydrogen bond may be the most fundamental interaction in biology and yet our understanding of and ability to predict the structures, energetics, and dynamics of hydrogen bonds and hydrogen bond networks are remarkably limited. In this research a quantitative and predictive model for hydrogen bond structure will be developed that focuses on factors that determine hydrogen bond length and coupling; hydrogen bond energetics within protein environments. The wide array of approaches utilized include site-directed mutagenesis and unnatural amino acid incorporation; protein structure and dynamics via NMR and cryo- and room temperature X-ray, and neutron crystallography; atomic-level energetics, dynamics, and catalysis via quantum mechanics/molecular mechanics (QM/MM); and assessment of small molecule hydrogen bond (H bond) energetics and ligand association via 1H NMR, isothermal titration calorimetry, small molecule neutron and X-ray diffraction, and quantum mechanics. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.