Determination of Long-Range Distances by Fast Magic-Angle-Spinning Radiofrequency-Driven ¹⁹F–¹⁹F Dipolar Recoupling NMR

Abstract

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 C-C and C-N 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 F, whose large gyromagnetic ratio in principle allows distances up to 2 nm to be measured. However,F possesses large chemical shift anisotropies (CSAs) as well as large isotropic chemical shift dispersions, which pose challenges to dipolar coupling measurements. Here, we demonstrate F-F distance measurements at high magnetic fields under fast magic-angle spinning (MAS) using radiofrequency-driven dipolar recoupling (RFDR). We show that F-F cross-peaks for distances up to 1 nm can be readily observed in two-dimensional F-F correlation spectra using less than 5 ms of RFDR mixing. This efficient F-F dipolar recoupling is achieved using practically accessible MAS frequencies of 15-55 kHz, moderate F radio frequency field strengths, and noH 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 F pulses. Fast MAS considerably reduces relaxation losses during the RFDR π-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%), theF 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 F RFDR approach is complementary toF 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.

Elucidating ligand-bound structures of membrane proteins using solid-state NMR spectroscopy

“…This cholesterol-M2 binding study exemplifies the versatility of solid-state NMR for determining the location and structure of lipophilic ligands to membrane proteins. Different NMR-sensitive nuclei provide complementary information: 13 C chemical shift is best for determining protein conformation, 2 H quadrupolar coupling probes molecular orientation, while sparsely incorporated 19 F is ideal for measuring intermolecular distances to ~2 nm [18,20,21].…”

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.…”

Heteronuclear and homonuclear radio-frequency-driven recoupling

“…Under control of radio frequency pulses, dipolar interactions can be switched on, or recoupled, in order to correlate nearby spins or to accurately determine internuclear distances. Recoupling sequences can be broadly categorized as homonuclear (Meier and Earl, 1986;Tycko and Dabbagh, 1990;Gullion and Vega, 1992;Bennett et al, 1992;Zhang et al, 2020;Gelenter et al, 2020;Takegoshi et al, 2001;Szeverenyi et al, 1982;Hou et al, 2011bHou et al, , 2013Carravetta et al, 2000;Bennett et al, 1998;Nielsen et al, 2011) or heteronuclear (Gelenter et al, 2020;Gullion and Schaefer, 1989;Jaroniec et al, 2002;Hing et al, 1992;Hartmann and Hahn, 1962;Rovnyak, 2008;Metz et al, 1994;Hediger et al, 1994;Hou et al, 2011a;Brinkmann and Levitt, 2001;Gelenter and Hong, 2018;Zhang et al, 2016;Nielsen et al, 2011).…”