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 (ν(R) = 62 kHz), where (13)C-(1)H 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.
Highlights
The full assignment of resonances and determination of molecular structure can be done by employing different multi-dimensional solid-state Nuclear Magnetic Resonance (NMR) protocols
In GB1 and light chain 8 (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
In a recent article employing small molecules as model samples, we have shown that a simple Cross Polarization with Variable Contact (CPVC) experiment performed under fast magic angle spinning (MAS) conditions is free from the discussed supra-limitations and provides very accurate values of hetero-nuclear (13C–1H, 15N–1H) dipolar couplings, D, which reflect the inter-atomic distances and/or molecular motions.[19]
Summary
The full assignment of resonances and determination of molecular structure can be done by employing different multi-dimensional solid-state NMR protocols. In the previous reports the 2D CPVC NMR approach was applied to study structural constraints in relatively simple model samples.[19,20] In these cases, very well resolved 13C isotropic signals have allowed unambiguous assignment of specific sites and analysis of C–H dipolar splittings.
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