Characterization of Macromolecular Dynamics Using Para-hydrogen Induced Polarization of Nuclear Spins
Grant
Overview
Affiliation
Other
View All
Overview
abstract
In this project funded by the Chemical Structure, Dynamics and Mechanisms A (CSDM-A) program of the Chemistry Division, Professor Christian Hilty of Texas A&M University and a team of graduate students are using nuclear magnetic resonance spectroscopy (NMR, a cousin of the magnetic resonance imaging used in medical technology) to investigate how proteins fold to attain their three-dimensional structure. The structure, structural changes and molecular interactions of proteins are responsible for their function in the most basic processes of life. While it is possible to determine the three-dimensional arrangement of atoms even in these large molecules, the measurements often are too slow to resolve changes that occur over time. Proteins normally embedded in the cell membrane pose additional challenges, because they depend on the interactions with lipid molecules even when they are purified for study. In this project, the investigators are overcoming some of these challenges by using a method called para-hydrogen induced polarization of nuclear spins, which enhances the signals of nuclear magnetic resonance and makes it possible to achieve these measurements more rapidly. With the application of these methods, the project contributes insights into the longstanding problem of membrane protein folding, of which many aspects remain unknown despite the knowledge of the folded, three-dimensional structures of the molecules involved. The graduate students involved in this research project are gaining knowledge and skills that will be valuable in their future career pursuits, be these in fundamental chemistry research or in the applications of NMR in chemical analysis, medicine, and other areas of science and technology.The project develops methods for the characterization of structural changes in non-equilibrium macromolecular processes, motivated by the goal to elucidate the mechanisms of folding and insertion of beta-barrel membrane proteins. The proven ability of NMR to provide chemical selectivity for the determination of structure and molecular dynamics is exploited. NMR spectroscopy is made compatible with the elucidation of these structures under non-equilibrium conditions through the use of hyperpolarization by para-hydrogen induced polarization. Hydrogen enriched in the para-spin state is delivered to biomolecular samples, to generate hyperpolarization of small molecules in-situ. A first aim demonstrates the use of this para-hydrogen enhancement for characterizing intermolecular interactions with the model protein dihydrofolate reductase. Multi-dimensional protein NMR experiments are developed that exploit the ability to regenerate and transfer hyperpolarization to the protein, and that enable measurement of the binding interface structure. In a second aim, catalysts for para-hydrogen polarization are designed, which are specifically compatible with the application to samples of biological macromolecules. Finally, in a third aim, NMR spectroscopy of thus hyperpolarized molecules is applied to the structural and kinetic characterization of the membrane insertion process of the beta-barrel proteins OmpX and OmpA, and of their interaction with chaperones. The methods developed in aims 1 and 2 provide the resolution necessary to follow these macromolecular processes on relevant time scales. In addition to the aforementioned student training in physical and biophysical chemistry and nuclear magnetic resonance s techniques, the educational component of this research project includes the development of low-field NMR instrumentation for undergraduate laboratories (and eventual public demonstrations), and enzyme catalysis themed experimental kits ("ChemBoxes") for K-12 students and teachers.This award reflects NSF''s statutory mission and has been deemed worthy of support through evaluation using the Foundation''s intellectual merit and broader impacts review criteria.
