Analysis of local molecular motions of aromatic sidechains in proteins by 2D and 3D fast MAS NMR spectroscopy and quantum mechanical calculations

Abstract

We report a new multidimensional magic angle spinning NMR methodology, which provides an accurate and detailed probe of molecular motions occurring on timescales of nano- to microseconds, in sidechains of proteins. The approach is based on a 3D CPVC-RFDR correlation experiment recorded under fast MAS conditions (νR = 62 kHz), where 13C-1H CPVC dipolar lineshapes are recorded in a chemical shift resolved manner. The power of the technique is demonstrated in model tripeptide Tyr-(D)Ala-Phe and two nanocrystalline proteins, GB1 and LC8. We demonstrate that, through numerical simulations of dipolar lineshapes of aromatic sidechains, their detailed dynamic profile, i.e., the motional modes, is obtained. In GB1 and LC8 the results unequivocally indicate that a number of aromatic residues are dynamic, and using quantum mechanical calculations, we correlate the molecular motions of aromatic groups to their local environment in the crystal lattice. The approach presented here is general and can be readily extended to other biological systems.

Improving dipolar recoupling for site-specific structural and dynamics studies in biosolids NMR: windowed RN-symmetry sequences

“…1 H- 13 C dipolar parameters can be conveniently measured in a 3D experiment, where the dipolar recoupling period is followed by a 13 C- 13 C mixing step, such as fpRFDR, as reported previously 32 and shown in Figure 1c in the context of wPARS recoupling sequence.…”

“…This result indicates that the fpRFDR mixing does not introduce any artifacts into the spectra, in accord with the previous findings for a 3D CPVC-RFDR experiment. 32 …”

Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies

“…The evidence of such dynamics under non-DNP conditions has been recently reported for aromatic sidechains. 63-67 In principle, analysis of the conformational space and the populations could provide a glimpse into the type of motions that exist. However, data at variable temperatures will be required for a quantitative analysis of such dynamics, which can depend on motional timescales, amplitudes, populations and potential energy barriers.…”

Fast magic-angle sample spinning solid-state NMR at 60–100 kHz for natural abundance samples

“…109,110,111,112,113,114 Its fast MAS variant allows to add 1 H-13 C dipolar dimensions in multidimensional experiments 68,115,116,117,118 Recently, it was demonstrated that on-resonance CW 14 N irradiation during the CSA recoupling period with rf field ~30 times smaller than the 14 N quadrupolar couplings largely decouples 14 N-1 H interactions leading to perfect 1 H CSA lineshapes 127 .…”