The role of intramolecular motion in biological function is widely accepted but not fully understood. Function depends on the time-averaged three-dimensional structure and time-dependent fluctuations from that structure. Thus, the incomplete picture of the mechanism by which time-dependent protein fluctuations couple to biological function, in particular enzyme catalysis, has limited the scientific progress in enzyme design and de novo protein engineering. This research project will address these deficiencies by integrating solution NMR relaxation dispersion experiments, X-ray crystallography, biochemical and biophysical experiments, and computational chemistry approaches to provide a model for the interplay of protein motion, structure and function. The NMR experiments will yield detailed, atomic resolution information on the enzyme motions that will be combined with biochemical experiments and site-directed mutagenesis to address function and the relationship between motion and function. The structural studies will provide a three-dimensional view of the enzyme with which to frame the dynamics and biochemical results and will be augmented by computational studies, which will allow modeling of higher energy conformational states and the mechanism of conformational motion.
In addition to the scientific merit, this project will impact other areas. The computational aspects of this research will involve collaboration with the laboratory of Professor Victor Batista. This partnership will enhance the infrastructure at Yale by fostering interactions between computational/theoretical scientists and experimentalists. Undergraduates from economically disadvantaged and rural areas of the United States typically do not have access to state-of-art scientific instrumentation or research. This project will continue to include these students in research activities during the summer months. The principal investigator is in contact with several colleges in West Virginia to identify interested students. Often students from these areas become science teachers. Broadening their science background will positively influence their future teaching ability. In addition, the principal investigator actively includes high school students in his research, in accord with Yale policy on 'minors in the laboratory'. In addition to advancing scientific discovery, the pairing of undergraduates and high school students with a graduate student in the principal investigator's lab provides another benefit as it exposes the graduate student to teaching, outside of the normal TA assignment thereby enhancing their teaching and training opportunities. Original research resulting from this project will be published in a timely fashion. Furthermore, the principal investigator will continue to publish review articles of these NMR methods and applications for a general, non-NMR audience. These types of publications have the potential to impact other fields and allow incorporation of new ideas into research programs for which NMR may not have been considered.
Enzymes catalyze chemical reactions that are essential for life. Clarifying the details of how enzymes function is crucial for advancement in chemistry, biology, and biotechnology. The work carried out under this NSF grant demonstrated the importance of concerted motions in the enzyme Ribonuclease A (RNase A) in its catalytic cycle. These studies, using Nuclear Magnetic Resonance (NMR) showed that regions of the enzyme, far from the active site where chemistry occurs, were crucial for allowing the enzyme to achieve its active conformation. The motion of a flexible loop located 20 Angstroms from the RNase A active stie was shown to facilitate the action of this enzyme. Moreover, alteration of the natural motion of this loop detrimentally impacted catalysis in RNase A. This work clearly demonstrated how small magnitude conformational motions that take place in about 1 millisecond can have a profound impact of the abiltiy of enzymes to catalyze reactions. This work lays a critical foundation for future studies on this important biochemical problem. In addition, in the process of carrying out this work scientists (including postdoctoral scholars, graduate students, and undergraduates) were trained in modern state-of-the-art biophysical techniques. Furthermore, in an effort to disseminate these results to a wide audience these findings were presented in peer-reviewd publications, at national and international conferences, and conveyed to local high school and grade school students to present a broad picture of scientific research.
