Diffusion and Conformational Dynamics of Semiflexible Macromolecules and Supramolecular Assemblies on Lipid Membranes

Understanding the interaction of polyelectrolytes with oppositely charged lipid membranes is an important issue of soft matter physics, which provides an insight into mechanisms of interactions between biological macromolecules and cell membranes. Despite the fact that many (bio)macromolecules and filamentous supramolecular assemblies show semiflexible behavior, prior to this work very little was known about the conformational dynamics and Brownian motion of semiflexible particles attached to freestanding lipid membranes. In order to address these issues, diffusion and conformational dynamics of semiflexible DNA molecules and filamentous fd-virus particles electrostatically adsorbed to cationic freestanding lipid membranes were studied on the single particle level by means of optical wide-field fluorescence microscopy. Supergiant unilamellar vesicles (SGUVs) with diameters larger than 100 μm represent a perfect model of a freestanding membrane. In this work, a method was developed that enabled the reliable and efficient electroformation of cationic SGUVs on ITO-coated coverslips. The utilization of SGUVs as model freestanding lipid bilayers allowed for determination of the previously unknown surface viscosity of DOPC/DOTAP membranes. In particular, the analysis of the translational diffusion coefficients of small (10, 20, 50 nm) membrane-attached anionic polystyrene beads has shown that the surface viscosity of DOPC/DOTAP membranes with CDOTAP = 1–7 mol% is independent of the DOTAP concentration and equals η = (5.9± 0.2)× 10−10 Pa s m. The fluorescence video-microscopy investigation of single DNA molecules attached to cationic SGUVs revealed a previously unreported conformational transition of a membranebound DNAmolecule from a 2D random coil, the original conformation in which DNA attaches to the membrane, to a compact globule. This membrane-mediated DNA condensation is favored at high cationic lipid concentrations in the membrane and long DNA contour lengths. The DNA compaction rate in the coil–globule transition is 124± 46 kbp/s, and the resulting DNA globule sizes were found to be 250–350 nm at DOPC membranes containing 1 mol% DOTAP and 130–200 nm for 7 mol% DOTAP, indicating a stronger compaction for higher charge densities in the membrane. Additional experiments with freestanding cationic membranes in the gel state and supported cationic lipid membranes with gel–fluid coexistence suggest that the DNA collapse on a freestanding fluid cationic membrane may be initiated by a local lipid segregation in the membrane and is accompanied by local membrane deformations, which eventually stabilize the compact DNA globule. Furthermore, in this work single molecule studies of random-coil DNA molecules and filamentous fd-virus particles on a freestanding cationic lipid bilayer with a low charge density were carried out. The experiments revealed that these particles can be described as semiflexible chains in 2D. Taken together, DNA molecules and fd-virus particles cover a broad range of the ratio of contour length and persistence length from 0.4 to 82. The results of this work demonstrate that the mobility of such membrane-attached semiflexible particles is strongly affected by hydrodynamics in the lipid membrane and the surrounding bulk fluid, and can in essence be described using a hydrodynamics-based theory for a disk-shaped solid membrane inclusion with a characteristic size approximately equal to the radii of gyration of the particles.