METHODOLOGY DEVELOPMENT FOR NMR STUDIES OF BIOMOLECULES

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The goal of this project is the development and evaluation of new, solution-state NMR methodologies for use in studies of the structure and dynamics of biomolecules. NMR spectroscopy is an extremely powerful tool for the study of biomolecules in solution, due to its ability to provide detailed information at an atomic level. The extent to which NMR spectroscopy can be exploited for the structure determination of biomolecules, the investigation of intermolecular interactions in systems such as drug/receptor enzyme/substrate and antibody/antigen complexes, and the characterization of biomolecular dynamics depends on the ability to manipulate the relevant NMR-active nuclei to yield the desired information. The enormous impact that NMR spectroscopy has made during the last decade in the investigation of biomolecules and biophysical processes is due largely to the continuing development of new or improved techniques for extracting useful data from the systems of interest. The proposal targets several general areas of solution-state NMR spectroscopy in which a number of specific goals will be pursued: 1) Improvements in methodology for resonance assignment will be sought. In particular, the development of enhanced sensitivity, relaxation- compensated techniques for performing coherence transfer experiments will be pursued. 2) Rotating-frame relaxation experiments for studying exchange processes in the microsecond to millisecond time regime will be investigated. The theoretical foundation for such experiments will be laid in order to open up new windows on biomolecular dynamics. Exchange effects in coherence transfer experiments will also be explored with the goal of designing more robust methods in the presence of exchange. 3) Heteronuclear cross-polarization experiments will be developed which will optimize sensitivity for several important applications. 4) Much excitement has been generated in the biomolecular NMR community over the recent introduction of higher field (750 MHz proton frequency) spectrometers. Significant improvements in spectral resolution and inherent sensitivity are anticipated for most nuclei. However, significant challenges will also be faced in realizing the fun potential of the new instrumentation. A thorough investigation will be conducted of various aspects of NMR spectroscopy at very high magnetic fields in the context of biomolecular NMR applications. 5) Novel experiments for measuring fast proton exchange rates and heteronuclear NOEs will be developed. The relative merits of single- versus triple-axis magnetic field gradients will be explored, and protocols will be investigated to facilitate the design, set-up and execution of optimized gradient- enhanced NMR experiments. 6) Our existing simulation software will be improved to allow a determination of nuclear spin dynamics under the influence of radio-frequency irradiation, spin relaxation and exchange processes. The correct interpretation of NMR data, and therefore the veracity of the resulting conclusions, depends critically on having not just some intuitive insight into the sophisticated NMR experiments employed but also a detailed understanding of the underlying physics.