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Design of amphiphilic protein maquettes: controlling assembly, membrane insertion, and cofactor interactions.

TitleDesign of amphiphilic protein maquettes: controlling assembly, membrane insertion, and cofactor interactions.
Publication TypeJournal Article
Year of Publication2005
AuthorsDischer, BM, Noy, D, Strzalka, J, Ye, S, Moser, CC, Lear, JD, J Blasie, K, P Dutton, L
JournalBiochemistry
Volume44
Issue37
Pagination12329-43
Date Published2005 Sep 20
ISSN0006-2960
KeywordsBinding Sites, Heme, Kinetics, Membrane Proteins, Models, Molecular, Molecular Sequence Data, Oxidation-Reduction, Peptides, Protein Structure, Secondary
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.

DOI10.1021/bi050695m
Alternate JournalBiochemistry
PubMed ID16156646
PubMed Central IDPMC2574520
Grant ListP01 GM048130-090007 / GM / NIGMS NIH HHS / United States
F32 GM063388-02 / GM / NIGMS NIH HHS / United States
P01 GM048130 / GM / NIGMS NIH HHS / United States
F32 GM063388-01 / GM / NIGMS NIH HHS / United States
GM48130 / GM / NIGMS NIH HHS / United States
GM63388 / GM / NIGMS NIH HHS / United States
P01 GM048130-110007 / GM / NIGMS NIH HHS / United States
P01 GM048130-100007 / GM / NIGMS NIH HHS / United States
F32 GM063388 / GM / NIGMS NIH HHS / United States