¹⁹F Dynamic Nuclear Polarization at Fast Magic Angle Spinning for NMR of HIV-1 Capsid Protein Assemblies

Abstract

We report remarkably high, up to 100-fold, signal enhancements in 19 F dynamic nuclear polarization (DNP) magic angle spinning (MAS) spectra at 14.1 T on HIV-1 CA capsid protein assemblies. These enhancements correspond to absolute sensitivity ratios of 12-29 and are of similar magnitude as seen for 1 H signals in the same samples. At MAS frequencies above 20 kHz, it was possible to record 2D 19 F-13 C HETCOR spectra, which contain long-range intra-and intermolecular correlations. Such correlations provide unique distance restraints, inaccessible in conventional experiments without DNP for protein structure determination. Furthermore, systematic quantification of the DNP enhancements as a function of biradical concentration, MAS frequency, temperature, and microwave power is reported. Our work establishes the power of DNP-enhanced 19 F MAS NMR spectroscopy for structural characterization of HIV-1 CA assemblies and this approach is anticipated to be applicable to a wide range of large biomolecular systems.

Structure and dynamics of the drug-bound bacterial transporter EmrE in lipid bilayers

“…These four components vary in intensities from 6% to 50% of the full spectral intensity, thus cannot be simply attributed to each of four fluorines. When the 19 F intensity was transferred from protein carbons by cross polarization (CP) 38,39 , the −103 ppm, −105 ppm, and −110 ppm peaks are preferentially enhanced, indicating that these resonances arise from fluorines that lie in close proximity to the protein carbons. The −110 ppm signal shows the largest intensity increase, indicating that this peak results from a 19 F (ppm) 13 C- 19 fluorine that is both structurally ordered and the closest to the protein.…”

The Potential of 19F NMR Application in GPCR Biased Drug Discovery

“…Therefore, NMR exhibits undeniable advantages in that the wild-type construct is used; the full energy landscape of the receptor conformation is maintained and can be presented at one time in a reconstitution system such as maltose-neopentyl glycol (MNG)-3 [62], nanodisc, or lipid bilayer systems [20,63,64]. On-going innovation of solid-state NMR technology, especially the dynamic nuclear polarization (DNP) technique [65] and its application in 19 F NMR [66], holds tremendous potential for profiling the receptor conformation faithfully in a genuine, native membrane environment or even in a living cell.…”

Visualizing HIV-1 Capsid and Its Interactions with Antivirals and Host Factors

“…NMR experiments allow for examination of the protein dynamics that are thought to contribute to the construction and pleomorphism of the HIV-1 capsid. MAS NMR, in particular, has improved considerably for large protein assemblies in recent years, demonstrated with the utility of both 19F MAS NMR, whereby 19F is substituted into the indole ring of a tryptophan residue in CA, and dynamic nuclear polarization (DNP) MAS NMR, in which signal intensity is increased by the addition of a polarizing agent [ 46 , 47 , 48 ].…”