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Water uptake in vesicles and the subsequent exchange between water protons and amide -NH protons in amino acids can be followed by a new, highly sensitive, type of magnetic resonance spectroscopy: dynamic nuclear polarisation (DNP)-enhanced NMR in the liquid state. Water hydrogen atoms are detected prior to and after their transfer to molecular sites in peptides and proteins featuring highly-accessible proton-exchangeable groups, as is the case for the -NH groups of intrinsically disordered proteins. The detected rates for amide proton-water proton exchange can be modulated by membrane-crossing rates, when a membrane channel is interposed. We hyperpolarised water proton spins via dynamic nuclear polarisation followed by sample dissolution (d-DNP) and transferred the created polarisation to -NH groups with high solvent accessibility in an intrinsically disordered protein domain. This domain is the membrane anchor of c-Src kinase, whose activity controls cell proliferation. The hindrance of effective water proton transfer rate constants observed in free solvent when a membrane-crossing step is involved is discussed. This study aims to assess the feasibility of recently-introduced hyperpolarised (DNP-enhanced) NMR to assess water membrane crossing dynamics.

We introduce a new symmetry-based method for structural investigations of areas surrounding water-exchanging hydrogens in biomolecules by liquid-state nuclear magnetic resonance spectroscopy. Native structures of peptides and proteins can be solved by NMR with fair resolution, with the notable exception of labile hydrogen sites. The reason why biomolecular structures often remain elusive around exchangeable protons is that the dynamics of their exchange with the solvent hampers the observation of their signals. The new spectroscopic method we report allows to locate water-originating hydrogens in peptides and proteins via their effect on nuclear magnetic transitions similar to electronic phosphorescence, long-lived coherences. The sign of long-lived coherences excited in coupled protons can be switched by the experimenter. The different effect of water-exchanging hydrogens on long-lived coherences with opposed signs allows to pinpoint the position of these labile hydrogen atoms in the molecular framework of peptides and proteins. [ABSTRACT FROM AUTHOR]