Orientation of the pore-forming peptide GALA in POPC vesicles determined by a BODIPY-avidin/biotin binding assay.
We determined the orientation of a biotinylated version of the pore-forming peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA) at pH 5.0 in large unilamellar phosphatidylcholine vesicles, using the enhancement of BODIPY-avidin fluorescence subsequent to its irreversible binding to a biotin moiety. GALA and its variants were biotinylated at the N- or C-terminus. BODIPY-avidin was either added externally or was pre-encapsulated in vesicles to assess the fraction of liposome-bound biotinylated GALA that exposed its labeled terminus to the external or internal side of the bilayer, respectively. Under conditions where most of the membrane-bound peptides were involved in transmembrane aggregates and formed aqueous pores (at a lipid/bound peptide molar ratio of 2500/1), the head-to-tail (N- to C-terminus) orientation of the membrane-inserted peptides was such that 3/4 of the peptides exposed their N-terminus on the inside of the vesicle and their C-terminus on the outside. Under conditions resulting in reduced pore formation (at higher lipid/peptide molar ratios), we observed an increase in the fraction of GALA termini exposed to the outside of the vesicle. These results are consistent with a model (Parente et al., Biochemistry, 29:8720, 1990) that requires a critical number of peptides (M) in an aggregate to form a transbilayer structure. When the peptides form an aggregate of size i, with i < M = 4 to 6, the orientation of the peptides is mostly parallel to the membrane surface, such that both termini of the biotinylated peptide are exposed to external BODIPY-avidin. This BODIPY-avidin/biotin binding assay should be useful to determine the orientation of other membrane-interacting molecules.
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University of California, School of Pharmacy, Departments of Biopharmaceutics and Pharmaceutical Chemistry, San Francisco, California 94143-0446, USA.
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D000595:Amino Acid Sequence; D001360:Avidin; D055592:Biophysical Phenomena; D001703:Biophysics; D001710:Biotin; D001896:Boron Compounds; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry*; D011485:Protein Binding
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008081:Liposomes; D010455:Peptides; D010713:Phosphatidylcholines; C066951:GALA peptide; D001360:Avidin; D001710:Biotin; C028694:1-palmitoyl-2-oleoylphosphatidylcholine