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 C-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 N andC. Here we present a robust 2D C-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 C-F REDOR sequence is combined with 2D C-C correlation to resolve multiple distances in highly C-labeled proteins. We show that, at the high magnetic fields which are important for obtaining well resolvedC spectra, the deleterious effect of the large 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 2DC-C resolved C-F REDOR technique on C,N-labeled GB1. A 5-F-Trp tagged GB1 sample shows the extraction of distances to a single fluorine atom, while a 3-F-Tyr labeled GB1 sample allows us to evaluate the effects of multi-spin coupling and statistical 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 2DC-F REDOR technique should facilitate SSNMR-based protein structure determination by increasing the measurable distances to the ~ 10 Å range.

Atomic structures of closed and open influenza B M2 proton channel reveal the conduction mechanism

“…Mixing of fluorinated protein with 13 C-labeled protein does not affect the protein conformation, as verified by identical 13 C chemical shifts (data not shown). Two 13 C spectra were measured without (S 0 ) and with (S) 19 F pulses using the rotational-echo double-resonance (REDOR) experiment 35 , 36 . To ensure that the distance-dependent dipolar couplings are not averaged by motion, we measured the REDOR spectra in the gel-phase membrane where the protein is immobilized.…”

“…Powder averaging used the REPULSION320 scheme 59 with 64 γ angles. The REDOR simulations accounted for finite 19 F and 13 C 180° pulse lengths, 19 F pulse imperfections, and 19 F chemical shift anisotropy (CSA) 36 . The 19 F pulse imperfection was treated by adding REDOR curves for 19 F pulse flip angles of 180° to 145° using weights that match a normal distribution centered at 180° with a standard deviation of 15° 36 .…”

Fast Magic‐Angle Spinning 19F NMR Spectroscopy of HIV‐1 Capsid Protein Assemblies

“…Only three recent studies used faster spinning frequencies. In two, resolution enhancements were observed at 35 and 40 kHz, [14, 26] while a report from our group demonstrated that frequencies of 40–60 kHz yielded significant line narrowing for fluorosubstituted tryptophan solids, thus alleviating the need for 1 H decoupling. [27] …”

Fully hydrophobic HIV gp41 adopts a hemifusion-like conformation in phospholipid bilayers

“…To further test the hemifusion structural model, it will be important to obtain additional long-range distance restraints. These may be obtained from paramagnetically tagged protein or fluorinated protein by exploiting paramagnetic relaxation enhancement or 19 F distance NMR techniques (32)(33)(34)(35), respectively. Finally, mixed labeled protein samples will be important to determine the oligomeric structure of this gp41 hemifusion intermediate.…”