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

Abstract

¹⁹ F NMR spectroscopy is an attractive and growing area of research with broad applications in biochemistry, chemical biology, medicinal chemistry, and materials science. We have explored fast magic angle spinning (MAS) ¹⁹ F solid-state NMR spectroscopy in assemblies of HIV-1 capsid protein. Tryptophan residues with fluorine substitution at the 5-position of the indole ring were used as the reporters. The ¹⁹ F chemical shifts for the five tryptophan residues are distinct, reflecting differences in their local environment. Spin-diffusion and radio-frequency-driven-recoupling experiments were performed at MAS frequencies of 35 kHz and 40-60 kHz, respectively. Fast MAS frequencies of 40-60 kHz are essential for consistently establishing ¹⁹ F-¹⁹ F correlations, yielding interatomic distances of the order of 20 Å. Our results demonstrate the potential of fast MAS ¹⁹ F NMR spectroscopy for structural analysis in large biological assemblies.

Keywords: 19F NMR spectroscopy; capsids; magic angle spinning; protein assemblies; protein structures.

Figure 1.

(a) Structures of the HIV-1 CA monomer (W23, W80, W117, W133 and W184 are shown in purple stick representation with fluorine atoms as yellow spheres), a section of the CA tube and the A14C/E45C/W184A/M185A CA cross-linked hexamer (NTD; grey, CTD; teal) (b) Transmission electron micrographs of U-¹⁵N CA tubes, 5-¹⁹F-Trp,U-¹⁵N tubes, 5-¹⁹F-Trp,U-¹³C,¹⁵N A14C/E45C/W184A/M185A tubes and 5F-Trp A14C/E45C/W184A/M185A CA soluble cross-linked hexamers. (c) ¹⁹F solution NMR spectra at 14.1 T of 5-¹⁹F-Trp,U-¹⁵N CA and 5-¹⁹F-Trp,U-¹³C,¹⁵N A14C/E45C/W184A/M185A CA cross-linked hexamer. (d) ¹⁹F MAS NMR spectra (19.96 T, MAS frequency; 40 kHz) of (top to bottom): 5-¹⁹F-Trp,U-¹³C,¹⁵N CA tubes, 5-¹⁹F-Trp,U-¹⁵N W23I mutant CA tubes, 5-¹⁹F-Trp,U-¹⁵N W80Y mutant CA tubes, 5-¹⁹F-Trp,U-¹³C,¹⁵N A14C/E45C/W184A/M185A mutant CA tubes, and A14C/E45C/W184A/M185A cross-linked CA hexamer precipitated with PEG-4000.

Figure 2.

¹⁹F MAS NMR spectra of 5-¹⁹F-Trp,U-¹⁵N CA tubes (11.74 T; MAS frequency 4 kHz (a), and 19.96 T; MAS frequency 15 kHz (b)). The inset in (a) shows the W23 resonance.

Figure 3.

2D ¹⁹F-¹⁹F correlation spectra (19.96 T) of (a) 5-¹⁹F-Trp CA tubes and (b) 5-¹⁹F-Trp A14C/E45C/W184A/M185A CA cross-linked hexamer tubes. MAS frequencies and mixing times in the ¹⁹F-¹⁹F RFDR spectra are listed. The first contour level was set to 5 x noise rmsd. No ¹H decoupling was applied. (c) Experimental ¹⁹F-¹⁹F RFDR buildup of the W80-W133 and W133-W80 cross-peak volumes in the 5-¹⁹F-Trp A14C/E45C/W184A/M185A CA cross-linked hexamer tubes (MAS frequency; 40 kHz). (d) Simulated ¹⁹F-¹⁹F RFDR cross-peak buildup curves for ¹⁹F-¹⁹F distances of 5–20 Å (simulation parameters are provided in the Supporting Information). The inset is an expansion for up to 200 ms.