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Rapid measurement of long-range distances in proteins by multidimensional 13C-19F 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 13C-15N 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 15N and 13C. Here we present a robust 2D 13C-19F 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 13C-19F REDOR sequence is combined with 2D 13C-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 13C-13C resolved 13C-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 13C-19F REDOR technique should facilitate SSNMR-based protein structure determination by increasing the measurable distances to the ~?10 Å range.

SUBMITTER: Shcherbakov AA

PROVIDER: S-EPMC6314655 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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

Shcherbakov Alexander A AA Hong Mei M

Journal of biomolecular NMR 20180521 1

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 13C-15N 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 15N and 13C ...[more]

PMID: 29785460

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Project description:Nanometer-range distances are important for restraining the three-dimensional structure and oligomeric assembly of proteins and other biological molecules. Solid-state NMR determination of protein structures typically utilizes 13C-13C and 13C-15N distance restraints, which can only be measured up to ?7 Å because of the low gyromagnetic ratios of these nuclear spins. To extend the distance reach of NMR, one can harvest the power of 19F, whose large gyromagnetic ratio in principle allows distances up to 2 nm to be measured. However, 19F possesses large chemical shift anisotropies (CSAs) as well as large isotropic chemical shift dispersions, which pose challenges to dipolar coupling measurements. Here, we demonstrate 19F-19F distance measurements at high magnetic fields under fast magic-angle spinning (MAS) using radiofrequency-driven dipolar recoupling (RFDR). We show that 19F-19F cross-peaks for distances up to 1 nm can be readily observed in two-dimensional 19F-19F correlation spectra using less than 5 ms of RFDR mixing. This efficient 19F-19F dipolar recoupling is achieved using practically accessible MAS frequencies of 15-55 kHz, moderate 19F radio frequency field strengths, and no 1H decoupling. Experiments and simulations show that the fastest polarization transfer for aromatic fluorines with the highest distance accuracy is achieved using either fast MAS (e.g., 60 kHz) with large pulse duty cycles (>50%) or slow MAS with strong 19F pulses. Fast MAS considerably reduces relaxation losses during the RFDR ?-pulse train, making finite-pulse RFDR under fast-MAS the method of choice. Under intermediate MAS frequencies (25-40 kHz) and intermediate pulse duty cycles (15-30%), the 19F CSA tensor orientation has a quantifiable effect on the polarization transfer rate; thus, the RFDR buildup curves encode both distance and orientation information. At fast MAS, the impact of CSA orientation is minimized, allowing pure distance restraints to be extracted. We further investigate how relayed transfer and dipolar truncation in multifluorine environments affect polarization transfer. This fast-MAS 19F RFDR approach is complementary to 19F spin diffusion for distance measurements and will be the method of choice under high-field fast-MAS conditions that are increasingly important for protein structure determination by solid-state NMR.

Dataset Information

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

Publications

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

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