title: Solubilization of a membrane protein by combinatorial supercharging.
abstract: Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intramembrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intramembrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.
authors.affiliation: Department of Molecular Biology and Biochemistry and ‡Department of Chemistry, University of California , Irvine, California 92697, United States.
authors.forename: Agnes
authors.initials: A
authors.lastname: Hajduczki
authors.forename: Sudipta
authors.initials: S
authors.lastname: Majumdar
authors.forename: Marie
authors.initials: M
authors.lastname: Fricke
authors.forename: Isola A M
authors.initials: IA
authors.lastname: Brown
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Weiss
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D051242:Caveolin 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D015700:HIV Envelope Protein gp41 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D055672:Static Electricity
chemical_list: D051242:Caveolin 1; D015700:HIV Envelope Protein gp41; D008024:Ligands; D019151:Peptide Library; D011994:Recombinant Proteins