title: Hydrogen Exchange Mass Spectrometry of Related Proteins with Divergent Sequences: A Comparative Study of HIV-1 Nef Allelic Variants.
abstract: Hydrogen exchange mass spectrometry can be used to compare the conformation and dynamics of proteins that are similar in tertiary structure. If relative deuterium levels are measured, differences in sequence, deuterium forward- and back-exchange, peptide retention time, and protease digestion patterns all complicate the data analysis. We illustrate what can be learned from such data sets by analyzing five variants (Consensus G2E, SF2, NL4-3, ELI, and LTNP4) of the HIV-1 Nef protein, both alone and when bound to the human Hck SH3 domain. Regions with similar sequence could be compared between variants. Although much of the hydrogen exchange features were preserved across the five proteins, the kinetics of Nef binding to Hck SH3 were not the same. These observations may be related to biological function, particularly for ELI Nef where we also observed an impaired ability to downregulate CD4 surface presentation. The data illustrate some of the caveats that must be considered for comparison experiments and provide a framework for investigations of other protein relatives, families, and superfamilies with HX MS. Graphical Abstract ᅟ.
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, 02115, USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Wales
authors.affiliation: Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261, USA.
authors.forename: Jerrod A
authors.initials: JA
authors.lastname: Poe
authors.affiliation: Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261, USA.
authors.forename: Lori
authors.initials: L
authors.lastname: Emert-Sedlak
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, 02115, USA.|Genzyme Corporation, Framingham, MA, 01701-9322, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Morgan
authors.affiliation: Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261, USA.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Smithgall
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, 02115, USA. j.engen@neu.edu.
authors.forename: John R
authors.initials: JR
authors.lastname: Engen
mesh_terms: D000595:Amino Acid Sequence; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D006859:Hydrogen; D013058:Mass Spectrometry; D051572:Proto-Oncogene Proteins c-hck; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000235:genetics*; D018909:src Homology Domains
chemical_list: D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D006859:Hydrogen; D051572:Proto-Oncogene Proteins c-hck
keywords: Hck SH3; Protein conformation; Sequence conservation