Abstract
Hyperpolarized water can be a valuable aid in protein NMR, leading to amide group 1H polarizations that are orders of magnitude larger than their thermal counterparts. Suitable procedures can exploit this to deliver 2D 1H–15N correlations with good resolution and enhanced sensitivity. These enhancements depend on the exchange rates between the amides and the water, thereby yielding diagnostic information about solvent accessibility. This study applied this “HyperW” method to four proteins exhibiting a gamut of exchange behaviors: PhoA(350–471), an unfolded 122-residue fragment; barstar, a fully folded ribonuclease inhibitor; R17, a 13.3 kDa system possessing folded and unfolded forms under slow interconversion; and drkN SH3, a protein domain whose folded and unfolded forms interchange rapidly and with temperature-dependent population ratios. For PhoA4(350–471) HyperW sensitivity enhancements were ≥300×, as expected for an unfolded protein sequence. Though fully folded, barstar also exhibited substantial enhancements; these, however, were not uniform and, according to CLEANEX experiments, reflected the solvent-exposed residues. R17 showed the expected superposition of ≥100-fold enhancements for its unfolded form, coexisting with more modest enhancements for their folded counterparts. Unexpected, however, was the behavior of drkN SH3, for which HyperW enhanced the unfolded but, surprisingly, enhanced even more certain folded protein sites. These preferential enhancements were repeatedly and reproducibly observed. A number of explanations—including three-site exchange magnetization transfers between water and the unfolded and folded states; cross-correlated relaxation processes from hyperpolarized “structural” waters and labile side-chain protons; and the possibility that faster solvent exchange rates characterize certain folded sites over their unfolded counterparts—are considered to account for them.
Highlights
Nuclear magnetic resonance (NMR) plays an irreplaceable role in biophysical studies
Improving sensitivity and signal-to-noise ratio in NMR has been the focus of extensive efforts, including the use of hyperpolarization methods that can impart orders-of-magnitude sensitivity enhancements to a variety of solutions and solids.[1−5] Out of all methods for nuclear hyperpolarization, dissolution dynamic nuclear polarization (DNP) stands out in its generality to enhance the sensitivity of high-field solutionstate NMR and MRI measurements.[6−9] the ex situ nature of this approach where the sample is hyperpolarized in one magnet under cryogenic conditions and transferred as a liquid to another system for its eventual observation fails when it is attempted on large biomolecules subject to very fast low-field relaxation processes
HMQC NMR experiments, to the point of highlighting lowpopulated “invisible” states that would be hard to observe in equilibrium with their more populated states.[68,69]
Summary
NMR can tackle complex systems such as proteins in solution under native or near-physiological conditions, and provide information about the structures and dynamics of these systems with atomic resolution Despite this potential, NMR in general and NMR of large biomolecules in particular suffers from inherent sensitivity issues. (HyperW) NMR was recently introduced to overcome this limitation and enable the study of proteins and nucleic acids.[13] HyperW NMR relies on the fact that H2O’s protons can be hyperpolarized by dissolution DNP into the tens of percent, and if suitably handled their relaxation times can reach into the tens of seconds These protons, being labile, can spontaneously exchange with groups in biomolecules for instance, with amides in unfolded proteins or intrinsically disordered proteins/domains (IDPs/IDDs). More recently,[17] this method was used to achieve substantial enhancements for the Parkinson’s-disease-associated IDP α-
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