title: Role of helix nucleation in the kinetics of binding of mastoparan X to phospholipid bilayers.
abstract: Many antimicrobial peptides undergo a coil-to-helix transition upon binding to membranes. While this conformational transition is critical for function, little is known about the underlying mechanistic details. Here, we explore the membrane-mediated folding mechanism of an antimicrobial peptide, mastoparan X. Using stopped-flow fluorescence techniques in conjunction with a fluorescence resonance energy transfer (FRET) pair, p-cyanophenylalanine (donor) and tryptophan (acceptor), we were able to probe, albeit in an indirect manner, the membrane-mediated folding kinetics of this peptide. Our results show that the association of mastoparan X with model lipid vesicles proceeds with biphasic kinetics. The first step shows a large change in the FRET signal, indicating that the helix forms early in the time course of the interaction, while the second step where a further increase in tryptophan fluorescence is observed presumably reflects deeper insertion of the peptide into the bilayer. Additional kinetic studies on a double mutant of mastoparan X, designed to form a nucleation site for alpha-helix formation through coordination with a metal ion (e.g., Zn2+ or Ni2+), indicate that while the coil-to-helix transition occurs in the first step, it follows the rate-determining docking of the peptide onto the membrane surface. Taken together, these results indicate that the initial association of the peptide with the membrane occurs in a nonhelical conformation, which rapidly converts to a helical state within the anisotropic environment of the bilayer surface.
authors.affiliation: Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Jia
authors.initials: J
authors.lastname: Tang
authors.forename: Rachel S
authors.initials: RS
authors.lastname: Signarvic
authors.forename: William F
authors.initials: WF
authors.lastname: DeGrado
authors.forename: Feng
authors.initials: F
authors.lastname: Gai
mesh_terms: D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008051:Lipid Bilayers; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D008051:Lipid Bilayers; D010455:Peptides; D010743:Phospholipids; C071123:mastoparan X