Engineering protein dynamics and function using NMR, mutagenesis, and calorimetry

蛋白质在生物体中扮演着各种各样的角色，了解它们的功能对于了解生命的分子基础至关重要。在过去的50年里，已经有可能通过实验精确地确定蛋白质的三维结构。这些信息对于解释生物活性非常有价值，然而对蛋白质结构的静态描述忽略了这样一个事实，即蛋白质是高度灵活的分子。最近(在过去的15年中)发展了测量蛋白质动力学的高分辨率方法，有迹象表明结构波动可能在蛋白质功能中发挥核心作用。然而，蛋白质的化学结构、内部运动和生物活性之间的关系目前还不是很清楚。在拟议的研究中，将制定有意设计蛋白质动力学的指导方针，提供一个强大的新研究工具；此外，结果将是了解蛋白质动力学和功能之间关系的独特窗口。在这些研究中，三种具有代表性的蛋白质将发生大量突变：一种酶，以及两种功能是识别和结合目标分子的蛋白质。这些突变将被设计成破坏原子间的相互作用，这种相互作用可能会赋予蛋白质结构刚性，从而改变灵活性。将使用核磁共振光谱在原子分辨率下测量工程蛋白质的动力学，并将根据结合的动力学和热力学以及催化效率来量化它们的功能特性。通过分析突变的影响，将制定工程蛋白质动力学指南。通过比较大量的动力学和功能数据，可以确定蛋白质动力学和功能之间具有统计学意义的相关性。

Proteins perform an enormous variety of roles in living organisms and understanding how they function is critical to understanding the molecular basis of life. For the past 50 years it has been possible to experimentally determine the three-dimensional structures of proteins with atomic precision. This information has been invaluable for explaining biological activity, however a static description of protein structure ignores the fact that proteins are highly flexible molecules. High-resolution methods for measuring protein dynamics have been developed recently (over the last 15 years), and there are indications that structural fluctuations may play a central role in protein function. However the relationship between the chemical structures, internal motions, and biological activities of proteins are currently not well understood. Guidelines for deliberately engineering protein dynamics will be developed in the proposed research, providing a powerful new investigative tool; in addition, the results will represent a unique window into the relationship between protein dynamics and function. In these investigations, a large number of mutations will be made in three representative proteins: an enzyme, and two proteins whose functions are to recognize and bind to target molecules. The mutations will be designed to disrupt interatomic interactions that likely confer rigidity on the protein structure, thereby altering flexibility. The dynamics of the engineered proteins will be measured at atomic resolution using nuclear magnetic resonance spectroscopy, and their functional properties will be quantified in terms of the kinetics and thermodynamics of binding, and in terms of catalytic efficiency. Through analysis of the effects of mutations, guidelines for engineering protein dynamics will be developed. Comparisons among the large number of dynamical and functional data will allow statistically significant correlations between protein dynamics and function to be identified.