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. 2018 Dec 10;57(50):16375-16379.
doi: 10.1002/anie.201809060. Epub 2018 Oct 19.

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

Mingzhang Wang  1   2 , Manman Lu  1   2   3 , Matthew P Fritz  1   2 , Caitlin M Quinn  1 , In-Ja L Byeon  2   3 , Chang-Hyeock Byeon  2   3 , Jochem Struppe  4 , Werner Maas  4 , Angela M Gronenborn  2   3 , Tatyana Polenova  1   2
Affiliations

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

Mingzhang Wang et al. Angew Chem Int Ed Engl. .

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.

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Figures

Figure 1.
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.
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.
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.
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