Membrane topology of the colicin E1 channel using genetically encoded fluorescence

Biochemistry. 2011 Jun 7;50(22):4830-42. doi: 10.1021/bi101934e. Epub 2011 May 11.

Abstract

The membrane topology of the colicin E1 channel domain was studied by fluorescence resonance energy transfer (FRET). The FRET involved a genetically encoded fluorescent amino acid (coumarin) as the donor and a selectively labeled cysteine residue tethered with DABMI (4-(dimethylamino)phenylazophenyl-4'-maleimide) as the FRET acceptor. The fluorescent coumarin residue was incorporated into the protein via an orthogonal tRNA/aminoacyl-tRNA synthetase pair that allowed selective incorporation into any site within the colicin channel domain. Each variant harbored a stop (TAG) mutation for coumarin incorporation and a cysteine (TGT) mutation for DABMI attachment. Six interhelical distances within helices 1-6 were determined using FRET analysis for both the soluble and membrane-bound states. The FRET data showed large changes in the interhelical distances among helices 3-6 upon membrane association providing new insight into the membrane-bound structure of the channel domain. In general, the coumarin-DABMI FRET interhelical efficiencies decreased upon membrane binding, building upon the umbrella model for the colicin channel. A tentative model for the closed state of the channel domain was developed based on current and previously published FRET data. The model suggests circular arrangement of helices 1-7 in a clockwise direction from the extracellular side and membrane interfacial association of helices 1, 6, 7, and 10 around the central transmembrane hairpin formed by helices 8 and 9.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acyl-tRNA Synthetases
  • Colicins / chemistry*
  • Colicins / genetics
  • Colicins / metabolism
  • Coumarins / chemistry
  • Cysteine / metabolism
  • Escherichia coli / metabolism
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes / metabolism
  • Lipid Bilayers / chemistry
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Structure, Secondary
  • p-Dimethylaminoazobenzene / analogs & derivatives
  • p-Dimethylaminoazobenzene / chemistry
  • p-Dimethylaminoazobenzene / metabolism

Substances

  • Colicins
  • Coumarins
  • Fluorescent Dyes
  • Lipid Bilayers
  • 4-dimethylaminophenylazophenyl-4'-maleimide
  • p-Dimethylaminoazobenzene
  • coumarin
  • Amino Acyl-tRNA Synthetases
  • Cysteine