Development of LED-Assisted NMR Technologies for the Atomic-Resolution Analysis of Medically Relevant Biomolecules in Solution at Submicromolar Concentration

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Project Summary The goal of this R01 renewal application is to further develop the LED-enhanced NMR technology (LC-photo- CIDNP) that was established during the prior cycle of funding, and extend the method to the facile and ultra- rapid 1D-to-3D NMR spectroscopy of proteins at sub-micromolar concentration. We will focus on NMR studies in solution and will target folded, unfolded and intrinsically disordered proteins in either buffered solution or cell- like media. We will accomplish the above goals within three steps. First (Specific Aim #1), we will incorporate a tryptophan (Trp) isotopolog bearing a quasi-isolated 1H-13C spin pair (QISP) within soluble proteins to achieve unprecedented NMR sensitivity for the detection of solvent-exposed Trp in proteins at nanomolar and sub-nanomolar levels. We will then employ the above technology in combination with field-cycling to achieve further NMR sensitivity enhancements. Second (Specific Aim #2) we will extend LC-photo-CIDNP to amino acids other than Trp and Tyr within proteins. This goal will be accomplished via through-space and through- bond polarization transfer methodologies. Third (Specific Aim #3), we will extend LC-photo-CIDNP to higher- dimensionality (>2D) NMR spectroscopy by developing novel 3D (and possibly 4D) 1H,13C heteronuclear spectroscopy pulse sequences tailored to the analysis of side-chain and backbone 1H-13C resonance pairs. This effort will include non-uniform-sampling (NUS) data collection schemes. We will then combine theoretical calculations and experiments to develop better LC-photo-CIDNP dyes with optimized g-factor values and long photoexcited-state lifetimes, for optimal LC-photo-CIDNP data collection. We will also exploit the peculiar field dependence of LED-enhanced NMR and implement 2D LC-photo-CIDNP on benchtop NMR spectrometers. Finally, we will test the success of the improved LC-photo-CIDNP technologies developed in this work by studying the interaction of an aggregation-prone client protein (SH3 variant) with the Hsp70 molecular chaperone at sub-micromolar concentration.