Phase transition behaviors of the supported DPPC bilayer investigated by sum frequency generation (SFG) vibrational spectroscopy and atomic force microscopy (AFM)

Abstract

The phase transition behaviors of a supported bilayer of dipalmitoylphosphatidylcholine (DPPC) have been systematically evaluated by in situ sum frequency generation (SFG) vibrational spectroscopy and atomic force microscopy (AFM). By using an asymmetric bilayer composed of per-deuterated and per-protonated monolayers, i.e., DPPC-d75/ DPPC and symmetric bilayer of DPPC/DPPC, we were able to probe the molecular structural changes during the phase transition process of the lipid bilayer by SFG spectroscopy. It was found that the DPPC bilayer is sequentially melted from the top (adjacent to the solution) to bottom leaflet (adjacent to the substrate) over a wide temperature range. The conformational ordering of the supported bilayer does not decrease (even slightly increases) during the phase transition process. The conformational defects in the bilayer can be removed after the complete melting process. The phase transition enthalpy for the bottom leaflet was found to be approximately three times greater than that for the top leaflet, indicating a strong interaction of the lipids with the substrate. The present SFG and AFM observations revealed similar temperature dependent profiles. Based on these results, the temperature-induced structural changes in the supported lipid bilayer during its phase transition process are discussed in a comparison with previous studies.

1H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers

“…33,37−46 To investigate the influence of defects on flip-flop, we performed random-walk (Monte Carlo) simulations of lateral lipid diffusion in the presence of 100 nm-diameter holes, a typical size observed in AFM images. 46 Figure 4a shows a schematic illustration of such a defect (not to scale) in a supported bilayer. We assumed that translocation effectively occurs by unhindered lateral diffusion of a lipid through the pore formed by the lipid headgroups.…”

“…On the other hand, AFM images of SSB generally show numerous patches of bare substrates appearing as holes with submicron dimensions (tens to hundreds of nanometers in diameter); these are especially ubiquitous in gel-phase bilayers. 33,37−46 Moreover, for typical values of bilayer surface coverage, the average distance between holes is on the order of microns or less. 46 Although these defects are often too small to be seen with conventional fluorescence microscopy, they can be detected with other fluorescence techniques.…”

“…33,37−46 Moreover, for typical values of bilayer surface coverage, the average distance between holes is on the order of microns or less. 46 Although these defects are often too small to be seen with conventional fluorescence microscopy, they can be detected with other fluorescence techniques. For example, in LB/LS supported bilayers containing fluorescent NBD-labeled lipids in both leaflets, external addition of the reducing agent dithionite—which does not permeate intact bilayers 66 —nevertheless quenched the fluorescence on both sides of the supported bilayer, implying the existence of numerous small defects in SSB that could not otherwise be visualized with fluorescence microscopy.…”

Phase Transitions in a Single Supported Phospholipid Bilayer: Real-Time Determination by Neutron Reflectometry

“…Phase transitions in single SLBs have been addressed mainly by means of AFM (atomic force microscopy) [11][12][13][14], SFG-VS (sum-frequency generation vibrational spectroscopy) [14], MD (molecular dynamics) simulations [15], and calorimetry [16]. The main common result is a substantial broadening of the gel-to-fluid phase transition (PT) with respect to that observed in freestanding bilayers in solution.…”

“…The main common result is a substantial broadening of the gel-to-fluid phase transition (PT) with respect to that observed in freestanding bilayers in solution. In some cases, AFM and calorimetry measurements have shown a decoupling between the PT behavior of the portion of the SLB facing the solid substrate (proximal leaflet) and the one closer to the water subphase (distal leaflet) [13,14,16]. In particular, Xie and co-workers [11] reported that the gel-to-fluid PT in a single DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) SLB is characterized by the formation and subsequent growth of fluid domains as the temperature is increased; coexistence between gel and fluid phases was observed in the central part of the PT, until the growing domains entirely replaced the starting phase.…”