Assessing site-specific enhancements imparted by hyperpolarized water in folded and unfolded proteins by 2D HMQC NMR.
Related Articles Assessing site-specific enhancements imparted by hyperpolarized water in folded and unfolded proteins by 2D HMQC NMR.
J Am Chem Soc. 2020 Apr 27;:
Authors: Szekely O, Olsen GL, Novakovic M, Rosenzweig R, Frydman L
Abstract
Dissolution DNP of hyperpolarized water can be a valuable aid in biomolecular NMR. With suitable optimizations one can utilize it to achieve, under near-physiological conditions, amide group 1H polarizations that are orders of magnitude larger than their thermal counterparts. Suitable experimental procedures can exploit this hyperpolarization to deliver 2D 1H-15N NMR correlations, with good site resolution and enhanced sensitivity. The resulting signal enhancements depend on the exchange rates between the amides and the water protons, thereby yielding diagnostic information about the former's solvent accessibility. This study applied the "HyperW" NMR method to four proteins, which exhibit a gamut of exchange behaviors. These included PhoA(350-471), an unfolded 122-residue fragment of Alkaline Phosphatase from E. coli; barstar, a fully folded ribonuclease inhibitor from Bacillus amyloliquefaciens; R17, a 13.3 kDa system possessing folded and unfolded forms under slow interconversion; and drkN SH3, an N-terminal signal transduction protein domain whose folded and unfolded forms interchange more rapidly and with temperature-dependent population ratios. For the disordered PhoA4(350-471) fragment 2D HyperW sensitivity enhancements were very high, >=300× over their thermal counterparts; this was expected due to the fast amide proton exchanges that occur throughout this unfolded protein sequence. Though fully folded, barstar also exhibited substantially-enhanced residues; these, however, were not uniform, and reflected what appeared well-folded but solvent-exposed residues, as confirmed by ancillary CLEANEX experiments. R17 showed in turn the expected superposition of >=100-fold enhancements for its unfolded form, coexisting with more modest enhancement for the folded counterparts. Unexpected, however, was the behavior of the drkN SH3 fragment, for which HyperW substantially enhanced both folded and unfolded states -but foremost of all certain sites of the folded protein. These preferential enhancements of folded over unfolded amide signals were repeatedly and reproducibly observed and a number of explanations - including three-site exchange magnetization transfers between water protons and amide protons from the unfolded and folded states, the possibility that faster rates of solvent exchange characterize the folded sites than their unfolded counterparts, and cross-correlated relaxation processes from hyperpolarized "structural" waters and labile sidechain protons that bias the enhancements towards the folded state- were considered to account for them.
PMID: 32338002 [PubMed - as supplied by publisher]
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