Rapid measurement of long-range distances in proteins by multidimensional 13 Câ?? 19 F REDOR NMR under fast magic-angle spinning
 

Rapid measurement of long-range distances in proteins by multidimensional 13 Câ?? 19 F REDOR NMR under fast magic-angle spinning

Abstract

The ability to simultaneously measure many long-range distances is critical to efficient and accurate determination of protein structures by solid-state NMR (SSNMR). So far, the most common distance constraints for proteins are 13Câ??15N distances, which are usually measured using the rotational-echo double-resonance (REDOR) technique. However, these measurements are restricted to distances of up to ~â??5Â*Ã? due to the low gyromagnetic ratios of 15N and 13C. Here we present a robust 2D 13Câ??19F REDOR experiment to measure multiple distances to ~â??10Â*Ã?. The technique targets proteins that contain a small number of recombinantly or synthetically incorporated fluorines. The 13Câ??19F REDOR sequence is combined with 2D 13Câ??13C correlation to resolve multiple distances in highly 13C-labeled proteins. We show that, at the high magnetic fields which are important for obtaining well resolved 13C spectra, the deleterious effect of the large 19F chemical shift anisotropy for REDOR is ameliorated by fast magic-angle spinning and is further taken into account in numerical simulations. We demonstrate this 2D 13Câ??13C resolved 13Câ??19F REDOR technique on 13C, 15N-labeled GB1. A 5-19F-Trp tagged GB1 sample shows the extraction of distances to a single fluorine atom, while a 3-19F-Tyr labeled GB1 sample allows us to evaluate the effects of multi-spin coupling and statistical 19F labeling on distance measurement. Finally, we apply this 2D REDOR experiment to membrane-bound influenza B M2 transmembrane peptide, and show that the distance between the proton-selective histidine residue and the gating tryptophan residue differs from the distances in the solution NMR structure of detergent-bound BM2. This 2D 13Câ??19F REDOR technique should facilitate SSNMR-based protein structure determination by increasing the measurable distances to the ~â??10Â*Ã? range.



Source: Journal of Biomolecular NMR