Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid Vesicles

© 2019 Biophysical Society 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. Nonenzymatic 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 functions, 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, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine 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 extent of membrane deformation. 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

Factors affecting patterns of tick parasitism on forest rodents in tick-borne encephalitis risk areas, Germany

Identifying factors affecting individual vector burdens is essential for understanding infectious disease systems. Drawing upon data of a rodent monitoring programme conducted in nine different forest patches in southern Hesse, Germany, we developed models which predict tick (Ixodes spp. and Dermacentor spp.) burdens on two rodent species Apodemus flavicollis and Myodes glareolus. Models for the two rodent species were broadly similar but differed in some aspects. Patterns of Ixodes spp. burdens were influenced by extrinsic factors such as season, unexplained spatial variation (both species), relative humidity and vegetation cover (A. flavicollis). We found support for the ‘body mass’ (tick burdens increase with body mass/age) and for the ‘dilution’ hypothesis (tick burdens decline with increasing rodent densities) and little support for the ‘sex-bias’ hypothesis (both species). Surprisingly, roe deer densities were not correlated with larvae counts on rodents. Factors influencing the mean burden did not significantly explain the observed dispersion of tick counts. Co-feeding aggregations, which are essential for tick-borne disease transmission, were mainly found in A. flavicollis of high body mass trapped in areas with fast increase in spring temperatures. Locally, Dermacentor spp. appears to be an important parasite on A. flavicollis and M. glareolus. Dermacentor spp. was rather confined to areas with higher average temperatures during the vegetation period. Nymphs of Dermacentor spp. mainly fed on M. glareolus and were seldom found on A. flavicollis. Whereas Ixodes spp. is the dominant tick genus in woodlands of our study area, the distribution and epidemiological role of Dermacentor spp. should be monitored closely