multifunctional-micropipette-based-method-for-incorporation

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

University of Maryland

Diphytanoylphosphatidyl choline (DPhPC) is a synthetic ester lipid with methylated tails found in archaeal ether lipids. Because of the stability of DPhPC bilayers and the absence of phase transitions over a broad range of temperatures, the lipid is used as an artificial membrane matrix for the reconstitution of channels, pumps, and membrane-active peptides. We characterized monomolecular films made of DPhPC and its natural ether analog DOPhPC at the air-water interface. We measured compression isotherms and dipole potentials of films made of DPhPC, DPhPE, and DOPhPC. We determined that at 40 mN/m the molecular area of DPhPC is 81.2 Å(2), consistent with X-ray and neutron scattering data obtained in liposomes. This indicates that 40 mN/m is the monolayer-bilayer equivalence pressure for this lipid. At this packing density, the compressibility modulus (Cs(-1 )= 122 ± 7 mN/m) and interfacial dipole potential (V = 355 ± 16 mV) were near their maximums. The molecular dipole moment was estimated to be 0.64 ± 0.02 D. The ether DOPhPC compacted to 70.4 Å(2)/lipid at 40 mN/m displaying a peak compressibility similar to that of DPhPC. The maximal dipole potential of the ether lipid was about half of that for DPhPC at this density, and the elemental dipole moment was about a quarter. The spreading of DPhPC and DOPhPC liposomes reduced the surface tension of the aqueous phase by 46 and 49 mN/m, respectively. This corresponds well to the monolayer collapse pressure. The equilibration time shortened as the temperature increased from 20 to 60 °C, but the surface pressure at equilibrium did not change. The data illustrates the properties of branched chains and the contributions of ester bonds in setting the mechanical and electrostatic parameters of diphytanoyl lipids. These properties determine an environment in which reconstituted voltage- or mechano-activated proteins may function. Electrostatic properties are important in the preparation of asymmetric folded bilayers, whereas lateral compressibility defines the tension in mechanically stimulated droplet interface bilayers.

Properties of diphytanoyl phospholipids at the air-water interface.

Membrane permeability is a desired property in drug design, but there have been difficulties in quantifying the direct drug partitioning into native membranes. Platensimycin (PL) is a new promising antibiotic whose biosynthetic production is costly. Six dialkylamine analogs of PL were synthesized with identical pharmacophores but different side chains; five of them were found inactive. To address the possibility that their activity is limited by the permeation step, we calculated polarity, measured surface activity and the ability to insert into the phospholipid monolayers. The partitioning of PL and the analogs into the cytoplasmic membrane of E. coli was assessed by activation curve shifts of a re-engineered mechanosensitive channel, MscS, in patch-clamp experiments. Despite predicted differences in polarity, the affinities to lipid monolayers and native membranes were comparable for most of the analogs. For PL and the di-myrtenyl analog QD-11, both carrying bulky sidechains, the affinity for the native membrane was lower than for monolayers (half-membranes), signifying that intercalation must overcome the lateral pressure of the bilayer. We conclude that the biological activity among the studied PL analogs is unlikely to be limited by their membrane permeability. We also discuss the capacity of endogenous tension-activated channels to detect asymmetric partitioning of exogenous substances into the native bacterial membrane and the different contributions to the thermodynamic force which drives permeation.

Anthony Yasmann

Department of Biology

University of Maryland

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

Anthony Yasmann

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biology

University of Maryland

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

Department of Biology