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理解生命的分子基础至关重要。在过去的50年里，人们已经可以通过实验以原子精度确定蛋白质的三维结构。这些信息对于解释生物活性是非常宝贵的，但是对蛋白质结构的静态描述忽略了蛋白质是高度灵活的分子这一事实。最近（过去15年）开发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没有很好的理解。在拟议的研究中，将制定故意设计蛋白质动力学的指导方针，提供一个强大的新调查工具;此外，结果将代表蛋白质动力学和功能之间关系的独特窗口。在这些研究中，将在三种代表性蛋白质中进行大量突变：一种酶和两种蛋白质，其功能是识别和结合靶分子。这些突变将被设计为破坏原子间的相互作用，这些相互作用可能赋予蛋白质结构刚性，从而改变柔性。将使用核磁共振光谱以原子分辨率测量工程蛋白的动力学，并且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的指导方针。大量的动力学和功能数据之间的比较将允许蛋白质动力学和功能之间的统计学显着的相关性被确定。

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.