Dynamic Nuclear Polarization Enhanced MAS NMR for Structural Analysis of HIV-1 Protein Assemblies.
Related Articles Dynamic Nuclear Polarization Enhanced MAS NMR for Structural Analysis of HIV-1 Protein Assemblies.
J Phys Chem B. 2015 Dec 28;
Authors: Gupta R, Lu M, Hou G, Caporini MA, Rosay M, Maas WE, Struppe JO, Suiter CL, Ahn J, Byeon IL, Franks WT, Orwick-Rydmark M, Bertarello A, Oschkinat H, Lesage A, Pintacuda G, Gronenborn AM, Polenova TE
Abstract
Mature infectious HIV-1 virions contain conical capsids comprised of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20 - 64 fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4 - 18.8 T) and temperature (109 - 180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine sidechain conformers, unique intermolecular correlations across two CA molecules, and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR as an excellent tool for characterization of HIV-1 assemblies.
PMID: 26709853 [PubMed - as supplied by publisher]
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