Methanol Accelerates DMPC Flip-Flop and Transfer: A SANS Study on Lipid Dynamics

Abstract

Methanol is a common solubilizing agent used to study transmembrane proteins/peptides in biological and synthetic membranes. Using small angle neutron scattering and a strategic contrast-matching scheme, we show that methanol has a major impact on lipid dynamics. Under increasing methanol concentrations, isotopically distinct 1,2-dimyristoyl-sn-glycero-3-phosphocholine large unilamellar vesicle populations exhibit increased mixing. Specifically, 1,2-dimyristoyl-sn-glycero-3-phosphocholine transfer and flip-flop kinetics display linear and exponential rate enhancements, respectively. Ultimately, methanol is capable of influencing the structure-function relationship associated with bilayer composition (e.g., lipid asymmetry). The use of methanol as a carrier solvent, despite better simulating some biological conditions (e.g., antimicrobial attack), can help misconstrue lipid scrambling as the action of proteins or peptides, when in actuality it is a combination of solvent and biological agent. As bilayer compositional stability is crucial to cell survival and protein reconstitution, these results highlight the importance of methanol, and solvents in general, in biomembrane and proteolipid studies.

Figure 1

Schematic of the contrast matching scheme used. Vesicles composed solely of d-DMPC (d54-DMPC) and h-DMPC are placed together in a H₂O/D₂O (55/45) mixture, contrast matched to an neutron scattering length density equal to fully mixed vesicles of d-DMPC and h-DMPC. Over time, because of lipid exchange and flip-flop, intensity loss can be monitored as vesicles mix and near the contrast match point.

Figure 2

(a) SANS curve of d-DMPC and h-DMPC vesicles with 3% (v/v) d-methanol solvent. Periodic measurements were conducted at 37°C over 38 h. (b) Normalized contrast decay curves of increasing d-methanol presence; continuous lines indicate fitted curves used to derive flip-flop and lipid exchange rate constants. Each data point represents the normalized integrated intensity of a single SANS curve similar to those found in (a). (c) Plot of measured flip-flop and lipid exchange rate constants as a function of d-methanol percentage concentration. Solid lines represent curves of best fit.