Site-specific protein backbone deuterium ²H_α quadrupolar patterns by proton-detected quadruple-resonance 3D ²H_αc_αNH...

A novel deuterium-excited and proton-detected quadruple-resonance three-dimensional (3D) ²H_αc_αNH MAS nuclear magnetic resonance (NMR) method is presented to obtain site-specific ²H_α 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 ²H_α, then transfer deuterium polarization to its attached C_α, 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 ¹H-¹⁵N 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 ²H_αc_αNH 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 ²H_α quadrupolar couplings to proton-detection, extending our previous triple-resonance demonstrations. Distortion-free excitation and polarization transfer of ∼160-170 kHz ²H_α quadrupolar coupling were presented by using a deuterium RF strength of ∼20 kHz. From these ²H_α 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 ²H_α lineshape is sensitive. Moreover, site-specific ²H_α T₁ relaxation times were obtained for a proof of concept. This 3D ²H_αc_αNH NMR experiment has the potential to determine structure and dynamics of perdeuterated proteins by utilizing deuterium as a novel reporter.