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BIOSCIENCES

6 NIST CENTER FOR NEUTRON RESEARCH

Methanol accelerates DMPC flip-flop 

and transfer: A SANS study on lipid dynamics

M. H. L. Nguyen,1 M. DiPasquale,1 B. W. Rickeard,1 C. B. Stanley,2 E. G. Kelley,3 and D. Marquardt1

1 University of Windsor, Windsor, Ontario ON N9B 3P4, Canada

2 Oak Ridge National Laboratory, Oak Ridge, TN 37831

3 NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD 20899

L

ipid bilayers form the structural backbone of cellular 

membranes and possess marked lateral and transversal 

organization of lipids. This strict lipid organization has 

implications in vital cellular processes, including protein 

function and localization, vesicle fusion and budding, and 

apoptosis. Lipids undergo diffusive motions that can disrupt 

their carefully assembled organization. This study focused on 

lipid exchange (between bilayers) and transverse diffusion (lipid 

flip-flop) as the former pertains to how lipids arrive, remain, 

and leave cellular membranes, whereas the latter disrupts the 

maintained membrane asymmetry (i.e., the compositional 

difference between leaflets) found in cells. In essence, both 

dynamical actions are intrinsically linked to bilayers and their 

compositional stability. Here, we examined how the common 

organic solvent methanol impact these dynamics.

In this work, we applied very small angle neutron scattering 

(VSANS) to monitor lipid mixing of two distinct 1,2-dimyristoylsn-glycero-3-phosphocholine (DMPC) populations, one chain 

perdeuterated DMPC (d-DMPC) and the other fully protiated 

DMPC (h-DMPC), in the presence of an increasing deuterated 

methanol concentration. The measurements were achieved 

by setting the ratio of H2O and D2O (here 45 % D2O) such 

that the water solvent neutron scattering length density 

was matched to uniformly mixed d-DMPC/h-DMPC vesicles. 

Unmixed vesicles will thus display contrast versus the water 

solvent, resulting in heightened scattering intensity, whereas 

fully mixed samples will display scattering intensities akin to the 

solvent background (i.e., a flat and featureless curve). Thus, as 

h-DMPC and d-DMPC large unilamellar vesicles begin to mix via 

lipid monomers transferring within and between bilayers, the 

measured intensity will decay and eventually reach an intensity 

baseline, corresponding to a single population of completely 

mixed vesicles (shown in Fig. 2a). Fig 1 illustrates such a strategy 

[1]. Further, a normalized intensity decay was calculated from the 

collective scattering curves of each sample and analyzed with 

a model for exchange/flip-flop (Fig. 2b). With this experimental 

setup, we quantified both DMPC flip-flop (kf) and exchange (ke) 

rates under the influence of methanol.

Increasing methanol concentrations had a profound effect 

on the kinetics of DMPC monomers in free-floating vesicles. 

Despite differences in vesicle size and investigative techniques, 

our unperturbed DMPC flip-flop and transfer rates agree and 

contrast previous findings. For example, Gerelli et al. used 

neutron reflectometry to measure DMPC flip-flop and exchange 

between vesicle dispersions and adsorbed planar bilayers [2]. 

Their exchange half-times for fluid-phase DMPC coincide with 

values found here (timescale of hours), whereas flip-flop was 

magnitudes faster (≤ 2.5 min). As recently shown however, 

the incomplete surface coverage of planar bilayers results in 

microscopic defects, which can facilitate lipid flip-flop and thus 

result in flip-flop rates on the order of seconds to minutes [3]. 

This study, with fully sealed vesicles, bypasses such issues. 

More significantly, our results reveal that methanol accelerates 

both fluid-phase DMPC flip-flop and transfer rates. The flipflop rate increases exponentially, whereas the exchange rate 

increases linearly under the studied concentrations (Fig. 2c). 

Methanol has by far the weakest hydrophobic character in the 

short-chain alcohol group yet seems to perturb the membrane 

through a similar fashion as longer chained alcohols and alkyl 

diols. Although it has been shown that other short-chain 

alcohols affect inward flipping rates, to our knowledge, we 

provide new insights with regards to both flip-flop and transfer 

rates in the presence of methanol. In general, at low methanol 

levels, DMPC undergoes slower flip-flop than transfer, but at 

methanol volume fractions greater than 2 %, the situation 

is reversed. Interestingly, these observations suggest that 

methanol affects the two dynamical processes in distinct ways. 

Dynamic light scattering (DLS) measurements of size and 

polydispersity were taken before and after incubation at multiple 

methanol concentrations, neither of which revealed significant 

changes, maintaining a vesicle diameter of 140 nm and a 

polydispersity index of 0.15 ± 0.02. Significantly, these results 

suggest fusion events did not occur as an increase in mean 

particle size and a polydispersity index would have been observed. 

Furthermore, SANS and small angle x-ray scattering (SAXS), were 

applied. SANS and SAXS are complementary techniques used to 

probe sample structure and are known to be extremely sensitive 

to membrane lamellarity and lipid bilayer structure. Both SANS 

and SAXS yielded curves that displayed diffuse scattering (i.e., no 

detectable sharp Bragg peak) demonstrating that multilamellar 

bilayers did not evolve under the presence of d-methanol. 

Collectively, DLS, SAXS and SANS indicate that methanol did not 
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iiNIST CENTER FOR NEUTRON RESEARCH FOREWORD aAf

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Foreword

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BIOSCIENCES

2019 ACCOMPLISHMENTS AND OPPORTUNITIES 7

alter the vesicles’ morphological structure at these concentrations.

To determine if a defect-mediated mechanism is responsible, 

we examined pertinent bilayer properties. Previous in silico 

studies revealed that area per lipid (AL) generally increases 

with increasing alcohol concentrations and this led to greater 

occurrences of bilayer defects [4]. Such defects can permit lipid 

headgroups to traverse the bilayer core and flip-flop. A joint 

refinement of SANS and SAXS data revealed that this was not 

the case; the scattering profiles of pure lipid and methanoltreated samples are near indiscernible. In terms of relevant 

bilayer structural parameters, the AL and bilayer thickness were 

essentially unchanged. Thus, the most likely explanation must 

involve methanol inducing short-range and perhaps short-lived 

defects, which are thus difficult to discern via methods (as 

those used here) that measure an ensemble structural average. 

Time-resolved SANS in combination with structural SANS, 

SAXS, and DLS measured the dynamical and structural 

effects of methanol on DMPC liposomes. Though structural 

deviations were not detected in the concentration regime 

studied, DMPC flip-flop and exchange were markedly 

enhanced. Our findings highlight an additional complication 

when externally adding biomolecules, whether it be protein, 

peptides or drugs. For example, in antimicrobial peptide 

(AMP) studies, though AMP attack is better simulated 

by external addition, these studies have the potential to 

incorrectly assign the cause of the enhanced lipid kinetics to 

the AMP. Ultimately, this work highlights the importance of 

understanding the interplay between the system of interest 

and the carrier solvent on lipid mobility whether it be model 

lipid systems or in vitro screening in cell biology.

FIGURE 1: Contrast matching strategy employed. Vesicles composed solely 

of d-DMPC d54-DMPC) and h-DMPC are placed together in a H2O/D2O (55/45) 

mixture with varying amounts of methanol, contrast matched to fully mixed 

vesicles of d-DMPC and h-DMPC. Over time, intensity loss can be monitored as 

vesicles mix (via exchange and flip-flop) and near the contrast match point.

FIGURE 2: (a) SANS curves 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 like those found in (a). (c) Plot of measured flip-flop and 

lipid exchange rate constants as a function of d-methanol volume percent. Solid 

lines represent curves of best fit.

References

[1] M. Nguyen, M. DiPasquale, B. Rickeard, C. Stanley, E. 

Kelley, D. Marquardt, Biophysical Journal, 116(5), 755 

(2019). 

[2] Y. Gerelli, L. Porcar, L. Lombardi, G. Fragneto, Langmuir, 

29(41), 12762 (2013).

[3] D. Marquardt, F. A. Heberle, T. Miti, B. Eicher, E. London, 

J. Katsaras, G. Pabst, Langmuir, 33(15), 3731 (2017). 

[4] H. V. Ly, M. L. Longo, Biophysical Journal, 87(2), 1013 

(2004).