Protein Hydrogen Exchange: Mechanism and Interpretation

University Of Pennsylvania, Philadelphia PA

Investigators

Abstract

Although hydrogen exchange (HX) has become the most powerful and fastest growing method for the study of molecular biophysics, the foundational knowledge on which the entire HX enterprise is based is still unfortunately insecure, which seriously hinders the interpretation of HX results. To secure those foundations, this project will pursue a wide ranging effort, integrating experimental and theoretical analysis, to examine the role of alternative HX mechanisms and the protein factors that determine HX behavior. Potential HX mechanisms include unhindered surface HX, solvent penetration, local fluctuations, cooperative subglobal and global unfolding. Potential protein factors include H-bonding, steric blocking, depth of burial, secondary structural type, electrostatic field, interaction density. The objective of this project is to obtain extensive site-resolved HX measurements under various test conditions by NMR techniques best suited for the intended HX time scale (Cleanex-PM for fast HX, standard HSQC for slower HX) and protein size (newly developed fast 3D NMR for larger proteins). In parallel, theoretical analyses will be used to study surface sterics, dynamics, and local fluctuational processes (MD and energy refinement), unfolding reactions (COREX), and electrostatic effects (DelPhi). Four proteins will be used for this study based on their special advantages (size, stability, high resolution structure, charge density, deep burial, known NMR time scale dynamics, preexisting HX information). In addition the large body of published HX data on other proteins provides a valuable reference data base. Examination of the important mechanisms and determining protein factors for many individual hydrogens will solidify HX knowledge and understanding, and will build toward a predictive capability. Broader Impacts The elaboration of HX mechanisms will provide a firm foundation for correctly interpreting the very many ongoing research studies that now use HX approaches as a major tool. Progress made will be communicated through publications in the research literature and in presentations at scientific meetings. Detailed data and results will be published on the laboratory web site for further analysis by the protein community. The scientific work planned in this project will continue to provide an excellent training experience for young scientists. In past years this laboratory has trained a large number of scientists who now hold positions at academic, governmental, and industrial centers. In this year four graduate students from across the University of Pennsylvania campus are training in this lab to enter research careers and this lab serves as a center for a number of others who share lab facilities and intellectual interactions. In past years three graduate students from this lab have been recognized for the Best PhD Dissertation of the Year at the University of Pennsylvania. In addition this lab has always supported a complement of high school and undergraduate college students and involved them in front line research. During the past year five high school students have participated in laboratory research projects. More broadly the current research of this laboratory informs the graduate level lecture/discussion course of the PI which this past year was ranked highest by student evaluation among 32 courses in the Biomedical Graduate Studies program at the U Penn School of Medicine. The PI chairs the Admissions Committee of the Biochemistry & Molecular Biophysics graduate program and has overseen an increasing entry of under represented minority students, including 9 URM applicants in the past two years. This project is jointly funded by Molecular Biophysics in the Division of Molecular and Cellular Bioscience and the Chemistry of Life Processes program in the Chemistry division.