Fast 19F Magic Angle Spinning NMR Crystallography for Structural Characterization of Fluorine-Containing Pharmaceutical Compounds.

Fluorine-containing compounds comprise 20 to 30 percent of all commercial drugs, and the proportion of fluorinated pharmaceuticals is rapidly growing. While magic angle spinning (MAS) NMR spectroscopy is a popular technique for analysis of solid pharmaceutical compounds, fluorine has been underutilized as a structural probe so far. Here, we report a fast (40-60 kHz) MAS 19F NMR approach for structural characterization of fluorine-containing crystalline pharmaceutical compounds at natural abundance, using the antimalarial fluorine-containing drug mefloquine as an example. We demonstrate the utility of 2D 19F-13C and 19F-19F dipolar-coupling-based correlation experiments for 19F and 13C resonance frequency assignment, which permit identification of crystallographically inequivalent sites. The efficiency of 19F-13C cross-polarization and the effect of 1H and 19F decoupling on spectral resolution and sensitivity were evaluated in a broad range of experimental conditions. We further demonstrate a protocol for measuring accurate interfluorine distances based on 1D DANTE-RFDR experiments combined with multispin numerical simulations.

Accuracy and precision of protein structures determined by magic angle spinning NMR spectroscopy: for some 'with a little help from a friend'.

We present a systematic investigation into the attainable accuracy and precision of protein structures determined by heteronuclear magic angle spinning solid-state NMR for a set of four proteins of varied size and secondary structure content. Structures were calculated using synthetically generated random sets of C-C distances up to 7 Å at different degrees of completeness. For single-domain proteins, 9-15 restraints per residue are sufficient to derive an accurate model structure, while maximum accuracy and precision are reached with over 15 restraints per residue. For multi-domain proteins and protein assemblies, additional information on domain orientations, quaternary structure and/or protein shape is needed. As demonstrated for the HIV-1 capsid protein assembly, this can be accomplished by integrating MAS NMR with cryoEM data. In all cases, inclusion of TALOS-derived backbone torsion angles improves the accuracy for small number of restraints, while no further increases are noted for restraint completeness above 40%. In contrast, inclusion of TALOS-derived torsion angle restraints consistently increases the precision of the structural ensemble at all degrees of distance restraint completeness.

Dynamic regulation of HIV-1 capsid interaction with the restriction factor TRIM5α identified by magic-angle spinning NMR and molecular dynamics simulations.

The host factor protein TRIM5α plays an important role in restricting the host range of HIV-1, interfering with the integrity of the HIV-1 capsid. TRIM5 triggers an antiviral innate immune response by functioning as a capsid pattern recognition receptor, although the precise mechanism by which the restriction is imposed is not completely understood. Here we used an integrated magic-angle spinning nuclear magnetic resonance and molecular dynamics simulations approach to characterize, at atomic resolution, the dynamics of the capsid's hexameric and pentameric building blocks, and the interactions with TRIM5α in the assembled capsid. Our data indicate that assemblies in the presence of the pentameric subunits are more rigid on the microsecond to millisecond timescales than tubes containing only hexamers. This feature may be of key importance for controlling the capsid's morphology and stability. In addition, we found that TRIM5α binding to capsid induces global rigidification and perturbs key intermolecular interfaces essential for higher-order capsid assembly, with structural and dynamic changes occurring throughout the entire CA polypeptide chain in the assembly, rather than being limited to a specific protein-protein interface. Taken together, our results suggest that TRIM5α uses several mechanisms to destabilize the capsid lattice, ultimately inducing its disassembly. Our findings add to a growing body of work indicating that dynamic allostery plays a pivotal role in capsid assembly and HIV-1 infectivity.

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

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.