Laser temperature jump experiments with fluorescence polarization and turbidity detection on the phase transition of single shell vesicles of dimyristoylphosphatidylcholine

Abstract

Abstract:The iodine-laser temperature-jump technique has been used to investigate the main phase transition in single shell vesicles of dimyristoylphosphatidylcholine, The probe molecules DPH and TMA-DPH were incorporated into the lipid bilayer and laser T-jump experiments with turbidity and flourescence polarization detection were performed. We found three well separated relaxation processes between 5 ~s and 10 ms. The relaxation signals showed strong cooperativity in the relaxation times as well as in their corresponding amplituedes. We attributed the relaxation to the formation and dissolution of clusters of different order inside the bilayer.

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

“…The experiments presented here reveal that the melting of lipid domains can occur on a 0.1-to 10-μs timescale, consistent with other IR experiments (6) and simulations (50). Global shape changes observed by turbidity experiments occur on a millisecond timescale (22), indicating a multistep mechanism to the gel-to-fluid transformation in vesicles. Local melting induced by the T-jump changes the thickness of the membrane, altering the hydrophobic mismatch between gD and the DMPC bilayer and inducing a conformational change in gD.…”

“…Membrane phase transitions are extremely complex. Light scattering (22), fluorescence (23), and IR spectroscopy (6) experiments have all revealed different elements of the process (summarized in Table S3). The timescales reported range from nanoseconds to greater than milliseconds, revealing that the transition is a multistage process.…”

“…However, they are consistent with the description of the phase transition as a multistep process. Speculatively, we may consider the similarity in timescales measured by turbidity change (22) to our millisecond component. These experiments are sensitive to size and shape changes of the vesicles.…”

“…2 A and B shows the kinetics for peak B of the ester groups for DMPC vesicles with and without gD over a 9°C range above T m : T f = T m + 1,6,10°C. Previous experiments on lipid vesicles, using a variety of probes, have found evidence of multiple relaxation timescales, and a dependence on the particular probe used (6,(22)(23)(24)(25). To address this uncertainty about the dynamics present, how they may depend on vibrational frequency, and which functional form is appropriate to describe the kinetics, we first used a maximum entropy (MaxEnt) method to determine the inverse Laplace transform (ILT) of the kinetic traces and obtain the underlying distribution of rates as a function, shown in Fig.…”

General discussion

“…For example, cholesterol appears to form a 1:l complex with lipid whereas each polypeptide molecule interacts with several nearby lipids (ca. [7][8][9][10] in each monolayer.…”