FCS Analysis of Protein Mobility on Lipid Monolayers

Abstract

In vitro membrane model systems are used to dissect complex biological phenomena under controlled unadulterated conditions. In this context, lipid monolayers are a powerful tool to particularly study the influence of lipid packing on the behavior of membrane proteins. Here, monolayers deposited in miniaturized fixed area-chambers, which require only minute amounts of protein, were used and shown to faithfully reproduce the characteristics of Langmuir monolayers. This assay is ideally suited to be combined with single-molecule sensitive fluorescence correlation spectroscopy (FCS) to characterize diffusion dynamics. Our results confirm the influence of lipid packing on lipid mobility and validate the use of FCS as an alternative to conventional surface pressure measurements for characterizing the monolayer. Furthermore, we demonstrate the effect of lipid density on the diffusional behavior of membrane-bound components. We exploit the sensitivity of FCS to characterize protein interactions with the lipid monolayer in a regime in which the monolayer physical properties are not altered. To demonstrate the potential of our approach, we analyzed the diffusion behavior of objects of different nature, ranging from a small peptide to a large DNA-based nanostructure. Moreover, in this work we quantify the surface viscosity of lipid monolayers. We present a detailed strategy for the conduction of point FCS experiments on lipid monolayers, which is the first step toward extensive studies of protein-monolayer interactions.

Control of Membrane Binding and Diffusion of Cholesteryl-Modified DNA Origami Nanostructures by DNA Spacers

“…It would be instructive to compare these results of diffusion of DNA nanostructures on lipid bilayers with their diffusion on lipid monolayers. Recently, we found the effective inclusion size of X5 with 18ds DNA spacers in low density 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) monolayers to be ≈28 nm, 75 which suggests that in this case, the DNA nanostructure is almost fully inserted into the lipid monolayer. This manifests the crucial difference in the molecular organization of lipid monolayers, featuring a relatively loose arrangement of lipids and bilayers, where lipid molecules are tightly packed together.…”

Patterning Microtubule Network Organization Reshapes Cell-Like Compartments

“…Lipids rapidly diffuse laterally within a lipid bilayer membrane and the lipid monolayer of an emulsion boundary. − Such rapid diffusion would quickly dissipate any induced spatial patterns of protein recruitment “painted” optically on the compartment boundary. Therefore, we sought to slow the diffusion of DGS-NTA­(Ni) lipid within the monolayer boundary by testing various lipids.…”

Fluorescent Submicron-Sized Poly(heptafluoro-n-butyl methacrylate) Particles with Long-Term Stability

“…The results obtained during DLS, NTA, and FCS measurements are presented in Table 3. The FCS method is typically used for size determination of small (single nanometers) fluorescent particles, such as dyes or proteins [37,38]. In the case of particles comparable in size to the size of the confocal volume, diffusion times measured by FCS are not proportional to the particle size.…”