Recruitment of Class I Hydrophobins to the Air:Water Interface Initiates a Multi-step Process of Functional Amyloid Formation

Abstract

Class I fungal hydrophobins form amphipathic monolayers composed of amyloid rodlets. This is a remarkable case of functional amyloid formation in that a hydrophobic:hydrophilic interface is required to trigger the self-assembly of the proteins. The mechanism of rodlet formation and the role of the interface in this process have not been well understood. Here, we have studied the effect of a range of additives, including ionic liquids, alcohols, and detergents, on rodlet formation by two class I hydrophobins, EAS and DewA. Although the conformation of the hydrophobins in these different solutions is not altered, we observe that the rate of rodlet formation is slowed as the surface tension of the solution is decreased, regardless of the nature of the additive. These results suggest that interface properties are of critical importance for the recruitment, alignment, and structural rearrangement of the amphipathic hydrophobin monomers. This work gives insight into the forces that drive macromolecular assembly of this unique family of proteins and allows us to propose a three-stage model for the interface-driven formation of rodlets.Class I hydrophobins are a family of small amphipathic proteins that are produced by filamentous fungi in a monomeric form but are able to self-assemble into amphipathic monolayers composed of amyloid-like structures known as rodlets (1-3). The polymerization of the hydrophobins occurs on contact with a hydrophobic:hydrophilic interface, such as an air:water boundary or when hydrophobins are secreted from the spores and come into contact with the air. Members of the hydrophobin family are characterized by the presence of four disulfide bonds. The amphipathic nature of hydrophobins drives them to the surface of solutions, where they reduce the surface tension (1).Some of the functional roles of the class I hydrophobins include acting as a surfactant at the air:water boundary to reduce the surface tension, which is a barrier to aerial growth of hyphae, and also to form a robust protein coat on spores. This coating provides a hydrophobic external surface that resists wetting and thus facilitates spore dispersal in air (4 -6). The class I hydrophobin rodlets share many of the structural characteristics of amyloid fibrils; formation of the insoluble, fibrillar rodlets is accompanied by conformational change to an ordered cross-␤-secondary structure form and the polymerized rodlets, but not the monomeric form of the protein, bind to the dye thioflavin T (ThT) 4 (2, 7, 8). However, in contrast to other amyloid fibrils, which can often be solubilized by treatment with denaturants such as guanidine hydrochloride or solvents such as dimethyl sulfoxide (9), treatment with acids such as formic and TFA has been reported to be the only method capable of depolymerizing hydrophobin rodlets and regenerating the monomeric form of the hydrophobin proteins in solution (10,11). This is similar to the curli and tafi fibrils produced by bacteria, which have been shown to be functional amyloid and which also req...

Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS

“…This does not happen spontaneously in solution, even at the high protein concentrations used for NMR structure determination, but requires association with a hydrophobic:hydrophilic interface. Surface tension is critical for triggering this conformational change, which may be analogous to the phenomenon of surface-induced denaturation of proteins (33) but is a functional requirement for Class I hydrophobins (34).…”

“…All NMR spectra were acquired, processed as described previously (34). Spectra were analyzed using Sparky (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco).…”

“…Fresh samples of proteins were prepared at 0.1 mg∕mL in 20% ethanol, or samples from ThT assembly assays were analyzed. Sample preparation and imaging were carried out as described previously (34).…”

Immobilization of LccC Laccase from Aspergillus nidulans on Hard Surfaces via Fungal Hydrophobins

“…In contrast, layers of class II hydrophobins can be easily dissolved by pressure, detergents, or ethanol. The assembly of class I hydrophobins in highly stable monolayers is associated with the formation of amyloid fibrillar structures and includes conformational changes of the protein molecules upon interaction (8,9).…”

Combined Effects of Agitation, Macromolecular Crowding, and Interfaces on Amyloidogenesis

“…Because of their amphiphilic character, these amyloid-forming peptides adsorb at an air-water interface (AWI; non-polar gas and polar aqueous solution) or more generally at hydrophobic-hydrophilic interfaces (HHI; gas-liquid, solid-liquid, or membranes) (9 -14). Adsorption to HHI allows these amyloid-forming peptides to spatially concentrate, to align their side chains with polar and non-polar side-chains segregating on opposite sides of the ␤-strand, and ultimately to promote their assembly into amyloid species (13)(14)(15)(16)(17)(18).…”