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

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