Doktorova, M. | Search bioRxiv

bioRxiv2018

Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Non-enzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels which have well-defined structures and known function, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small-angle X-ray scattering, circular dichroism and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time suggestive of scrambling activity, and the effect of the channel on the transbilayer lipid distribution in both symmetric POPC and asymmetric POPC/DMPC vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the amount of membrane deformation in the samples. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.

biophysics↗

bioRxiv2018

A new computational method for membrane compressibility: Bilayer mechanical thickness revisited

Because lipid bilayers can bend and stretch in ways similar to thin elastic sheets, physical models of bilayer deformation have utilized mechanical constants such as the moduli for bending rigidity ({kappa}C) and area compressibility (KA). However, the use of these models to quantify the energetics of membrane deformation associated with protein-membrane interactions and the membrane response to stress is often hampered by the shortage of experimental data suitable for the estimation of the mechanical constants of various lipid mixtures. While computational tools such as Molecular Dynamics (MD) simulations can provide alternative means to estimate KA values, current approaches suffer significant technical limitations. Here, we present a novel computational framework that allows for a direct estimation of KA values for individual bilayer leaflets. The theory is based on the concept of elasticity and derives KA from real-space analysis of local thickness fluctuations sampled in MD simulations. We explore and validate the model on a large set of single and multicomponent bilayers of different lipid composition and sizes, simulated at different temperatures. The calculated bilayer compressibility moduli agree with values estimated previously from experiments and those obtained from a standard computational method based on a series of constrained tension simulations. We further validate our framework in a comparison with an existing polymer brush model (PBM) and confirm the PBMs predicted linear relationship with proportionality coefficient of 24 using elastic parameters calculated from the simulation trajectories. The robustness of the results that emerge from the new method allows us to revisit the origins of the bilayer mechanical (compressible) thickness and in particular, its dependence on acyl chain unsaturation and the presence of cholesterol.

biophysics↗