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

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 C-13-N-15 distances, which are usually measured using the rotational-echo double-resonance (REDOR) technique. However, these measurements are restricted to distances of up to similar to 5 due to the low gyromagnetic ratios of N-15 and C-13. Here we present a robust 2D C-13-F-19 REDOR experiment to measure multiple distances to similar to 10 . The technique targets proteins that contain a small number of recombinantly or synthetically incorporated fluorines. The C-13-F-19 REDOR sequence is combined with 2D C-13-C-13 correlation to resolve multiple distances in highly C-13-labeled proteins. We show that, at the high magnetic fields which are important for obtaining well resolved C-13 spectra, the deleterious effect of the large F-19 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 C-13-C-13 resolved C-13-F-19 REDOR technique on C-13, N-15-labeled GB1. A 5-F-19-Trp tagged GB1 sample shows the extraction of distances to a single fluorine atom, while a 3-F-19-Tyr labeled GB1 sample allows us to evaluate the effects of multi-spin coupling and statistical F-19 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 C-13-F-19 REDOR technique should facilitate SSNMR-based protein structure determination by increasing the measurable distances to the similar to 10 range.