Glycophorin A Helical Transmembrane Domains Dimerize in Phospholipid Bilayers: A Resonance Energy Transfer Study

Abstract

Glycophorin A and its isolated transmembrane region (GpATM) are each known to form sequence-specific dimers in SDS micelles. Whether this behavior accurately reflects behavior in red cell membranes or lipid bilayers, however, has remained unclear. Resonance energy transfer between labeled GpATM peptides has been used to observe dimerization of GpATM in bilayers. Separate populations of GpATM peptides were labeled with 2,6-dansyl chloride as the donor chromophore and dabsyl chloride as the acceptor. Quenching of the 2,6-dansyl chloride by the dabsyl group demonstrated an association of the labeled peptides. The quenching was not affected by increases in the amount of lipid present or by unlabeled heterologous peptides but was decreased by the addition of unlabeled GpATM. GpATM was determined to form dimers by fitting the observed energy transfer for a number of donor to acceptor ratios and fitting to the expected number of donor labeled peptides in an oligomer with an acceptor as a function of oligomer number. The finding that the GpATM peptide forms helical dimers in lipid bilayers supports the idea that GpA is a dimer in the erythrocyte membrane. The resonance energy transfer approach may extend to the study of other oligomeric complexes.

Helix Induction in Antimicrobial Peptides by Alginate in Biofilms

“…3a, the emission maximum is quenched when the dabsyl-labeled Phe peptide was added to the dansyl-labeled peptide in alginate solution. Similar to results with the glycophorin dimer, quenching was not affected over a range of 5-fold increase in the amount of alginate present but decreased upon the addition of unlabeled Phe peptide (results not shown), indicating that the FRET signal is not simply the result of nearby but noninteracting peptides (11). A nonlinear curve fit to the observed energy transfer for a number of donor-to-acceptor ratios indicated that the peptide likely forms dimers and/or higher oligomers in alginate solutions (results not shown).…”

“…This technique has been widely used to study interactions between subunits in protein complexes (19,20), e.g. where dansyl-chloride-labeled and dabsylchloride-labeled glycophorin A peptides in lipid vesicles were used to demonstrate that the protein exists in a dimeric form (11). Thus, two cationic antimicrobial peptides with guest res- (5).…”

“…The peptides consist of a nonamphipathic hydrophobic core sequence (11)(12)(13)(14)(15)(16)(17)(18)(19) residues) flanked at one or both termini by a number of Lys or Arg residues. These peptides, which were originally designed as transmembrane mimetic model peptides (6,7), have the prototypic sequence KKAAAXAAAAAX-AAWAAXAAAKKKK-NH 2 with several key features: (i) Ala is the preferred background residue because of its mid-range hydropathy and frequent occurrence in membrane protein transmembrane domains; (ii) a Trp residue is inserted into the hydrophobic segment as a fluorescent probe; and (iii) N-and C-terminal hydrophilic Lys residues act to solubilize the otherwise hydrophobic peptides in aqueous media to facilitate purification and characterization.…”

Use of thiol-disulfide equilibria to measure the energetics of assembly of transmembrane helices in phospholipid bilayers

“…Thus, to understand folding of membrane proteins it is essential to discover systems in which folding is in thermodynamic equilibrium and to develop methods to quantitatively assess this equilibrium in membrane-like environments. Several biophysical techniques have been intensively used in thermodynamic studies of membrane protein association in detergent systems (9)(10)(11)(12)(13)(14)(15). However, the ideal environment for studying membrane proteins is the lipid bilayer because lipids mimic more closely the native membrane environment.…”

Specificity in transmembrane helix–helix interactions can define a hierarchy of stability for sequence variants

“…The propensity of the glycophorin A transmembrane domain to dimerize in a sequence-specific manner has been a paradigm for study of transmembrane helix-helix association in hydrophobic environments (4)(5)(6)(7)(8)(9). An additional advantage for detailed thermodynamic analysis of the GpA transmembrane segment (TMS) dimerization is the fact that a solution NMR structure has been solved (10).…”