title: Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies.
abstract: Mature infectious HIV-1 virions contain conical capsids composed 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 the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain 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 spectroscopy as an excellent tool for the characterization of HIV-1 assemblies.
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware , Newark, Delaware 19716, United States.
authors.forename: Rupal
authors.initials: R
authors.lastname: Gupta
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware , Newark, Delaware 19716, United States.
authors.forename: Manman
authors.initials: M
authors.lastname: Lu
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware , Newark, Delaware 19716, United States.
authors.forename: Guangjin
authors.initials: G
authors.lastname: Hou
authors.affiliation: Bruker Biospin Corporation , 15 Fortune Drive, Billerica, Massachusetts United States.
authors.forename: Marc A
authors.initials: MA
authors.lastname: Caporini
authors.affiliation: Bruker Biospin Corporation , 15 Fortune Drive, Billerica, Massachusetts United States.
authors.forename: Melanie
authors.initials: M
authors.lastname: Rosay
authors.affiliation: Bruker Biospin Corporation , 15 Fortune Drive, Billerica, Massachusetts United States.
authors.forename: Werner
authors.initials: W
authors.lastname: Maas
authors.affiliation: Bruker Biospin Corporation , 15 Fortune Drive, Billerica, Massachusetts United States.
authors.forename: Jochem
authors.initials: J
authors.lastname: Struppe
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware , Newark, Delaware 19716, United States.
authors.forename: Christopher
authors.initials: C
authors.lastname: Suiter
authors.forename: Jinwoo
authors.initials: J
authors.lastname: Ahn
authors.forename: In-Ja L
authors.initials: IJ
authors.lastname: Byeon
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie , Robert-Roessle-Straße 10, 13125 Berlin, Germany.
authors.forename: W Trent
authors.initials: WT
authors.lastname: Franks
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie , Robert-Roessle-Straße 10, 13125 Berlin, Germany.
authors.forename: Marcella
authors.initials: M
authors.lastname: Orwick-Rydmark
authors.affiliation: Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques, UMR 5280 CNRS/Ecole Normale Supérieure de Lyon , 5 rue de la Doua, 69100 Villeurbanne (Lyon), France.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bertarello
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie , Robert-Roessle-Straße 10, 13125 Berlin, Germany.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Oschkinat
authors.affiliation: Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques, UMR 5280 CNRS/Ecole Normale Supérieure de Lyon , 5 rue de la Doua, 69100 Villeurbanne (Lyon), France.
authors.forename: Anne
authors.initials: A
authors.lastname: Lesage
authors.affiliation: Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques, UMR 5280 CNRS/Ecole Normale Supérieure de Lyon , 5 rue de la Doua, 69100 Villeurbanne (Lyon), France.
authors.forename: Guido
authors.initials: G
authors.lastname: Pintacuda
authors.forename: Angela M
authors.initials: AM
authors.lastname: Gronenborn
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware , Newark, Delaware 19716, United States.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Polenova
mesh_terms: D002213:Capsid / Q000737:chemistry; D015497:HIV-1 / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011487:Protein Conformation; D014764:Viral Proteins / Q000737:chemistry*
chemical_list: D014764:Viral Proteins