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.
This project was directed at advancing and applying hydrogen exchange (HX) methodology, a major technique for the study of protein structure, dynamics, interactions, and function. In initial work a large database of protein HX results was acquired using NMR to measure HX rates at amino acid resolution. The results were critically compared with values predicted by leading theoreticians and/or expected on the basis of other hypotheses about presumed structural and dynamic determinants of the protein HX process. Agreement with the theoretical calculations and with previous hypotheses was poor. We then compared our measured HX rates with various structural parameters known at high resolution for the proteins we studied. Results show that H-bonded sites cannot exchange but requires a dynamic structural excursion that transiently breaks the protecting H-bond. The details of particular dynamic distortions could be often be specified. They range from whole molecule unfolding, through smaller cooperative unfolding reactions of secondary structural elements, and down to local fluctuations that involve as little as a single peptide group or side chain or water molecule. The operative motion is determined by surrounding structure and not by nearness to solvent or the strength of the protecting H-bond itself or its acceptor type (main chain, side chain, structurally bound water). Results are detailed in two publications. Other work was directed at improving the hydrogen exchange – mass spectrometry (HX MS method. We showed how to obtain hundreds of peptide fragments with four proteins ranging in size up to 906 amino acids, how to minimize back exchange during sample analysis, and we wrote two computer programs that supports the technology. One called ExMS makes it possible to efficiently process crowded mass spectra and definitively identify and characterize these many peptide fragments. Another called HDsite is able to analyze HX MS results to amino acid resolution. These results are described in four publications. We applied our advanced HX MS methods to study structure and dynamics in apolioprotein A-I, the major protein responsible for HDL metabolism. We studied apoA-I when free in solution, when bound to discoidal and to globular cholesterol-rich lipid particles, and also two interesting naturally occurring mutant forms. These results have broad interest for studies of the factors that promote and militate against heart disease. These results are described in four publications.
