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

点击翻译按钮获取中文摘要

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.