title: Design of amphiphilic protein maquettes: controlling assembly, membrane insertion, and cofactor interactions.
abstract: We have designed polypeptides combining selected lipophilic (LP) and hydrophilic (HP) sequences that assemble into amphiphilic (AP) alpha-helical bundles to reproduce key structure characteristics and functional elements of natural membrane proteins. The principal AP maquette (AP1) developed here joins 14 residues of a heme binding sequence from a structured diheme-four-alpha-helical bundle (HP1), with 24 residues of a membrane-spanning LP domain from the natural four-alpha-helical M2 channel of the influenza virus, through a flexible linking sequence (GGNG) to make a 42 amino acid peptide. The individual AP1 helices (without connecting loops) assemble in detergent into four-alpha-helical bundles as observed by analytical ultracentrifugation. The helices are oriented parallel as indicated by interactions typical of adjacent hemes. AP1 orients vectorially at nonpolar-polar interfaces and readily incorporates into phospholipid vesicles with >97% efficiency, although most probably without vectorial bias. Mono- and diheme-AP1 in membranes enhance functional elements well established in related HP analogues. These include strong redox charge coupling of heme with interior glutamates and internal electric field effects eliciting a remarkable 160 mV splitting of the redox potentials of adjacent hemes that leads to differential heme binding affinities. The AP maquette variants, AP2 and AP3, removed heme-ligating histidines from the HP domain and included heme-ligating histidines in LP domains by selecting the b(H) heme binding sequence from the membrane-spanning d-helix of respiratory cytochrome bc(1). These represent the first examples of AP maquettes with heme and bacteriochlorophyll binding sites located within the LP domains.
authors.affiliation: Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. bohdana@mail.med.upenn.edu
authors.forename: Bohdana M
authors.initials: BM
authors.lastname: Discher
authors.forename: Dror
authors.initials: D
authors.lastname: Noy
authors.forename: Joseph
authors.initials: J
authors.lastname: Strzalka
authors.forename: Shixin
authors.initials: S
authors.lastname: Ye
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Moser
authors.forename: James D
authors.initials: JD
authors.lastname: Lear
authors.forename: J Kent
authors.initials: JK
authors.lastname: Blasie
authors.forename: P Leslie
authors.initials: PL
authors.lastname: Dutton
mesh_terms: D001665:Binding Sites; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D017433:Protein Structure, Secondary
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D006418:Heme