Design of Amphiphilic Protein Maquettes: Controlling Assembly, Membrane Insertion, and Cofactor Interactions
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{sub H} heme binding sequence from the membrane-spanning d-helix of respiratory cytochrome bc{sub 1}. These represent the first examples of AP maquettes with heme and bacteriochlorophyll binding sites located within the LP domains.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 913843
- Report Number(s):
- BNL-78411-2007-JA; BICHAW; TRN: US200804%%237
- Journal Information:
- Biochemistry, Vol. 44, Issue 37; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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