Self-Assembled Hydrophobin Protein Films at the Air−Water Interface:  Structural Analysis and Molecular Engineering

Abstract

Hydrophobins are amphiphilic proteins produced by filamentous fungi. They function in a variety of roles that involve interfacial interactions, as in growth through the air-water interface, adhesion to surfaces, and formation of coatings on various fungal structures. In this work, we have studied the formation of films of the class II hydrophobin HFBI from Trichoderma reesei at the air-water interface. Analysis of hydrophobin aqueous solution drops showed that a protein film is formed at the air-water interface. This elastic film was clearly visible, and it appeared to cause the drops to take unusual shapes. Because adhesion and formation of coatings are important biological functions for hydrophobins, a closer structural analysis of the film was made. The method involved picking up the surface film onto a solid substrate and imaging the surface by atomic force microscopy. High-resolution images were obtained showing both the hydrophilic and hydrophobic sides of the film at nanometer resolution. It was found that the hydrophobin film had a highly ordered structure. To study the orientation of molecules and to obtain further insight in film formation, we made variants of HFBI that could be site specifically conjugated. We then used the avidin-biotin interaction as a probe. On the basis of this work, we suggest that the unusual interfacial properties of this type of hydrophobins are due to specific molecular interactions which lead to an ordered network of proteins in the surface films that have a thickness of only one molecule. The interactions between the proteins in the network are likely to be responsible for the unusual surface elasticity of the hydrophobin film.

Hydrophobin as a Nanolayer Primer That Enables the Fluorinated Coating of Poorly Reactive Polymer Surfaces

“…Most importantly, a discrete portion of their exposed surface is composed of amino acids with hydrophobic sidechains; this hydrophobic patch endows hydrophobins with exceptional amphiphilic properties and drives their spontaneous and rapid self-assembly at hydrophobic/hydrophilic interfaces, such as air/water and oil/water boundaries, where they pack into ordered structures and form remarkably strong and elastic fi lms. [ 22,23 ] Hydrophobins also effi ciently assemble onto solid surfaces to form amphiphilic fi lms that are able to reverse the surface wettability of the coated material [ 24 ] and allow for the immobilization of, e.g., proteins or enzymes onto solid surfaces [25][26][27][28] while preserving all of the features of the immobilized biomolecules. [ 29 ] The main advantage of this method is its simplicity: hydrophobin self-assembly proceeds spontaneously at room temperature within seconds or minutes and requires only a very tiny amount of protein.…”

The relation between solution association and surface activity of the hydrophobin HFBI from Trichoderma reesei

“…As surface active molecules, hydrophobins adsorb to the airwater interface and form monomolecular films that can have unusual visco-elastic properties [9]. Hydrophobins also form foams very easily [10,11] and adsorb to various surfaces [4,[12][13][14][15].…”

“…Hydrophobins also form foams very easily [10,11] and adsorb to various surfaces [4,[12][13][14][15]. At the air-water interface hydrophobins self-assemble into nanoscale structures, such as hexagonally ordered crystalline monolayers [9,16,17] or randomly aligned rodlets [18,19].…”

Fungal and mushroom hydrophobins: A review

“…Class I hydrophobins are highly insoluble in aqueous solution and can only be dissociated by concentrated strong acids, e.g. trifluoroacetic acid (TFA) and formic acid (Szilvay et al, 2007;Linder, 2009). Class I monolayer contains the same highly-ordered core structure known as rodlets, and is positive to Congo red and thioflavin T (Morris et al, 2012).…”