Site-specific protein backbone deuterium H quadrupolar patterns by proton-detected quadruple-resonance 3D HcNH MAS NMR spectroscopy.

A novel deuterium-excited and proton-detected quadruple-resonance three-dimensional (3D) HcNH MAS nuclear magnetic resonance (NMR) method is presented to obtain site-specific H deuterium quadrupolar couplings from protein backbone, as an extension to the 2D version of the experiment reported earlier. Proton-detection results in high sensitivity compared to the heteronuclei detection methods. Utilizing four independent radiofrequency (RF) channels (quadruple-resonance), we managed to excite the H, then transfer deuterium polarization to its attached C, followed by polarization transfers to the neighboring backbone nitrogen and then to the amide proton for detection. This experiment results in an easy to interpret HSQC-like 2D H-N fingerprint NMR spectrum, which contains site-specific deuterium quadrupolar patterns in the indirect third dimension. Provided that four-channel NMR probe technology is available, the setup of the HcNH experiment is relatively straightforward, by using low power deuterium excitation and polarization transfer schemes we have been developing. To our knowledge, this is the first demonstration of a quadruple-resonance MAS NMR experiment to link H quadrupolar couplings to proton-detection, extending our previous triple-resonance demonstrations. Distortion-free excitation and polarization transfer of ∼160-170 kHz H quadrupolar coupling were presented by using a deuterium RF strength of ∼20 kHz. From these H patterns, an average backbone order parameter of S = 0.92 was determined on a deuterated SH3 sample, with an average η = 0.22. These indicate that SH3 backbone represents sizable dynamics in the microsecond timescale where the H lineshape is sensitive. Moreover, site-specific H T relaxation times were obtained for a proof of concept. This 3D HcNH NMR experiment has the potential to determine structure and dynamics of perdeuterated proteins by utilizing deuterium as a novel reporter.