title: Mapping of protein:protein contact surfaces by hydrogen/deuterium exchange, followed by on-line high-performance liquid chromatography-electrospray ionization Fourier-transform ion-cyclotron-resonance mass analysis.
abstract: For protein complexes too large, uncrystallizable/insoluble, or low concentration for conventional X-ray diffraction or nuclear magnetic resonance analysis, the contact surface(s) may be mapped by comparing H/2H exchange rate (and thus solvent accessibility) of backbone amide hydrogens in free vs. complexed protein(s). The protein is first exposed to 2H2O, allowed to exchange for each of several reaction periods, and then digested with pepsin. The extent and rate of H/2H exchange is determined by measuring the increase in mass with H/2H exchange period for each of the peptides. Here, we present an experimental protocol that combines rapid (to minimize back-exchange) HPLC front-end separation with ultrahigh-resolution mass analysis (needed to distinguish the isotopic distributions of dozens of peptides simultaneously). The method is used to study the assembled human immunodeficiency virus type capsid protein (CA) and its soluble form.
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA.
authors.forename: TuKiet T
authors.initials: TT
authors.lastname: Lam
authors.forename: Jason K
authors.initials: JK
authors.lastname: Lanman
authors.forename: Mark R
authors.initials: MR
authors.lastname: Emmett
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Hendrickson
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
authors.forename: Peter E
authors.initials: PE
authors.lastname: Prevelige
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003903:Deuterium / Q000737:chemistry*; D019281:Dimerization; D005583:Fourier Analysis; D006859:Hydrogen / Q000737:chemistry*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D011994:Recombinant Proteins; D006859:Hydrogen; D003903:Deuterium