Submillisecond Dynamics of Mastoparan X Insertion into Lipid Membranes

Abstract

The mechanism of protein insertion into a lipid bilayer is poorly understood because the kinetics of this process is difficult to measure. We developed a new approach to study insertion of the antimicrobial peptide Mastoparan X into zwitterionic lipid vesicles, using a laser-induced temperature-jump to initiate insertion on the microsecond time scale and infrared and fluorescence spectroscopies to follow the kinetics. Infrared probes the desolvation of the peptide backbone and yields biphasic kinetics with relaxation lifetimes of 12 and 117 μs, whereas fluorescence probes the intrinsic tryptophan residue located near the N-terminus and yields a single exponential phase with a lifetime of 440 μs. Arrhenius analysis of the temperature-dependent rates yields an activation energy for insertion of 96 kJ/mol. These results demonstrate the complexity of the insertion process and provide mechanistic insight into the interplay between peptides and the lipid bilayer required for peptide transport across cellular membranes.

Binding, folding and insertion of a β-hairpin peptide at a lipid bilayer surface: Influence of electrostatics and lipid tail packing

“…The fluorescence signal is broader in the LUV than SUV as there is more scattering from the larger vesicles. The peptide buries more deeply into the fluid phase of the vesicles than the gel phase, a behavior analogous to that observed for the AMP Mastoparan X [53]. This insertion process is irreversible; once the peptide has buried into the membrane at high temperature, there is no red shift or change in intensity as the membrane returns to the low-temperature phase (See Figure S6, Supporting Information).…”

Time-resolved measurements of an ion channel conformational change driven by a membrane phase transition

“…This refers to domains of the membrane undergoing a transition to the fluid phase (described by APL, acyl chain disorder, and membrane thickness), but global changes in vesicle shape and curvature are not yet complete. This model is bolstered by the observation that the lipid phase-dependent insertion of a peptide into a membrane could be observed on a submillisecond timescale after a T-jump across the T m (28).…”

A photoswitchable helical peptide with light-controllable interface/transmembrane topology in lipidic membranes

“…An alternative approach has been to induce the TM insertion of a membrane-bound peptide by rapidly increasing the membrane fluidity with a laser-induced temperature-jump (T-jump) (Schuler et al, 2016). Although this allowed for (sub)microsecond resolution and sample conditions compatible with infrared (IR) spectroscopy, the changes in the membrane fluidity lasted for less than 1 ms (Schuler et al, 2016), making relevant millisecond and slower dynamical events inaccessible. Some of the limitations of fast mixing methods (Roder et al, 2006) and T-jumps (Kubelka, 2009) for the folding/ unfolding of water-soluble peptides have been avoided by their coupling to photoisomerizable organic molecules known as photoswitches (Blanco-Lomas et al, 2012;Hamm et al, 2008;Kumita et al, 2000).…”

“…However, the observed changes have been limited to a resolution of milliseconds and to single wavelength traces from Trp fluorescence spectroscopy, with limited information content. An alternative approach has been to induce the TM insertion of a membrane-bound peptide by rapidly increasing the membrane fluidity with a laser-induced temperature-jump (T-jump) ( Schuler et al., 2016 ). Although this allowed for (sub)microsecond resolution and sample conditions compatible with infrared (IR) spectroscopy, the changes in the membrane fluidity lasted for less than 1 ms ( Schuler et al., 2016 ), making relevant millisecond and slower dynamical events inaccessible.…”