title: Probing the paramyxovirus fusion (F) protein-refolding event from pre- to postfusion by oxidative footprinting.
abstract: To infect a cell, the Paramyxoviridae family of enveloped viruses relies on the coordinated action of a receptor-binding protein (variably HN, H, or G) and a more conserved metastable fusion protein (F) to effect membrane fusion and allow genomic transfer. Upon receptor binding, HN (H or G) triggers F to undergo an extensive refolding event to form a stable postfusion state. Little is known about the intermediate states of the F refolding process. Here, a soluble form of parainfluenza virus 5 F was triggered to refold using temperature and was footprinted along the refolding pathway using fast photochemical oxidation of proteins (FPOP). Localization of the oxidative label to solvent-exposed side chains was determined by high-resolution MS/MS. Globally, metastable prefusion F is oxidized more extensively than postfusion F, indicating that the prefusion state is more exposed to solvent and is more flexible. Among the first peptides to be oxidatively labeled after temperature-induced triggering is the hydrophobic fusion peptide. A comparison of peptide oxidation levels with the values of solvent-accessible surface area calculated from molecular dynamics simulations of available structural data reveals regions of the F protein that lie at the heart of its prefusion metastability. The strong correlation between the regions of F that experience greater-than-expected oxidative labeling and epitopes for neutralizing antibodies suggests that FPOP has a role in guiding the development of targeted therapeutics. Analysis of the residue levels of labeled F intermediates provides detailed insights into the mechanics of this critical refolding event.
authors.affiliation: Department of Molecular Biosciences and.
authors.forename: Taylor A
authors.initials: TA
authors.lastname: Poor
authors.affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202;
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Jones
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602;
authors.forename: Amika
authors.initials: A
authors.lastname: Sood
authors.affiliation: Department of Molecular Biosciences andHoward Hughes Medical Institute, Northwestern University, Evanston, IL 60208;
authors.forename: George P
authors.initials: GP
authors.lastname: Leser
authors.affiliation: Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130;
authors.forename: Manolo D
authors.initials: MD
authors.lastname: Plasencia
authors.affiliation: Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130;
authors.forename: Don L
authors.initials: DL
authors.lastname: Rempel
authors.affiliation: Department of Structural Biology, Stanford University, Stanford, CA 94305; and.
authors.forename: Theodore S
authors.initials: TS
authors.lastname: Jardetzky
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602;School of Chemistry, National University of Ireland, Galway, Ireland.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Woods
authors.affiliation: Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130;
authors.forename: Michael L
authors.initials: ML
authors.lastname: Gross
authors.affiliation: Department of Molecular Biosciences andHoward Hughes Medical Institute, Northwestern University, Evanston, IL 60208; ralamb@northwestern.edu.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Lamb
mesh_terms: D010084:Oxidation-Reduction; D045402:Parainfluenza Virus 5 / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D058849:Protein Refolding; D014760:Viral Fusion Proteins / Q000737:chemistry*
chemical_list: D010455:Peptides; D014760:Viral Fusion Proteins
keywords: mass spectroscopy; protein refolding; viral fusion protein