Adhesion Properties of Freestanding Hydrophobin Bilayers.

Hydrophobins are a family of small-sized proteins featuring a distinct hydrophobic patch on the protein's surface, rendering them amphiphilic. This particularity allows hydrophobins to self-assemble into monolayers at any hydrophilic/hydrophobic interface. Moreover, stable pure protein bilayers can be created from two interfacial hydrophobin monolayers by contacting either their hydrophobic or their hydrophilic sides. In this study, this is achieved via a microfluidic approach, in which also the bilayers' adhesion energy can be determined. This enables us to study the origin of the adhesion of hydrophobic and hydrophilic core bilayers made from the class II hydrophobins HFBI and HFBII. Using different fluid media in this setup and introducing genetically modified variants of the HFBI molecule, the different force contributions to the adhesion of the bilayer sheets are studied. It was found that in the hydrophilic contact situation, the adhesive interaction was higher than that in the hydrophobic contact situation and could be even enhanced by reducing the contributions of electrostatic interactions. This effect indicates that the van der Waals interaction is the dominant contribution that explains the stability of the observed bilayers.

Pure Protein Bilayers and Vesicles from Native Fungal Hydrophobins.

Pure protein bilayers and vesicles are formed using the native, fungal hydrophobin HFBI. Bilayers with hydrophobic (red) and hydrophilic (blue) core are produced and, depending on the type of core, vesicles in water, oily media, and even in air can be created using microfluidic jetting. Vesicles in water are even able to incorporate functional gramicidin A pores.

Fast membrane hemifusion via dewetting between lipid bilayers.

The behavior of lipid bilayers is important to understand the functionality of cells like the trafficking of ions. Standard procedures to explore the properties of lipid bilayers and hemifused states typically use supported membranes or vesicles. Both techniques have several shortcomings in terms of bio-relevance or accessibility for measurements. In this article, the formation of individual free standing hemifused states between model cell membranes is studied using an optimized microfluidic scheme which allows for simultaneous optical and electrophysiological measurements. In the first step, two model membranes are formed at a desired location within a microfluidic device using a variation of the droplet interface bilayer (DiB) technique. In the second step, the two model membranes are brought into contact forming a single hemifused state. For all tested lipids, the hemifused state between free standing membranes forms within hundreds of milliseconds, i.e. several orders of magnitude faster than those reported in literature. The formation of a hemifused state is observed as a two stage process, whereas the second stage can be explained as a dewetting process under no-slip boundary conditions. The formed hemifusion states have a long lifetime and a single fusion event can be observed when triggered by an applied electric field as demonstrated for monoolein.