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Hydrophobins are remarkable proteins due to their ability to self-assemble into amphipathic coatings that reverse surface wettability. Here, the versatility of the Class I hydrophobins EAS Δ15 and DewY in diverse nanosuspension and coating applications is demonstrated. The hydrophobins are shown to coat or emulsify a range of substrates including oil, hydrophobic drugs, and nanodiamonds and alter their solution and surface behavior. Surprisingly, while the coatings confer new properties, only a subset is found to be resistant to hot detergent treatment, a feature previously thought to be characteristic of the functional amyloid form of Class I hydrophobins. These results demonstrate that substrate surface properties can influence the molecular structures and physiochemical properties of hydrophobin and possibly other functional amyloids. Functional amyloid assembly with different substrates and conditions may be analogous to the propagation of different polymorphs of disease-associated amyloid fibrils with distinct structures, stability, and clinical phenotypes. Given that amyloid formation is not required for Class I hydrophobins to serve diverse applications, our findings open up new opportunities for their use in applications requiring a range of chemical and physical properties. In hydrophobin nanotechnological applications where high stability of assemblies is required, simultaneous structural and functional characterization should be carried out. Finally, while results in this study pertain to synthetic substrates, they raise the possibility that at least some members of the pseudo-Class I and Class III hydrophobins, reported to form assemblies with noncanonical properties, may be Class I hydrophobins adopting alternative structures in response to environmental cues.

The fungal hydrophobins are small proteins that are able to self-assemble spontaneously into amphipathic monolayers at hydrophobic:hydrophilic interfaces. These protein monolayers can reverse the wettability of a surface, making them suitable for increasing the biocompatibility of many hydrophobic nanomaterials. One subgroup of this family, the class I hydrophobins, forms monolayers that are composed of extremely robust amyloid-like fibrils, called rodlets. Here, we describe the protocols for the production and purification of recombinant hydrophobins and oxidative refolding to a biologically active, soluble, monomeric form. We describe methods to trigger the self-assembly into the fibrillar rodlet state and techniques to characterize the physicochemical properties of the polymeric forms.

In recent years, much attention has focused on incorporating biological and bio-inspired nanomaterials into various applications that range from functionalising surfaces and enhancing biomolecule binding properties, to coating drugs for improved bioavailability and delivery. Hydrophobin proteins, which can spontaneously assemble into amphipathic layers at hydrophobic:hydrophilic interfaces, are exciting candidates for use as nanomaterials. These unique proteins, which are only expressed by filamentous fungi, have been the focus of increasing interest from the biotechnology industry, as evidenced by the sharply growing number of hydrophobin-associated publications and patents. Here, we explore the contribution of different hydrophobins to supporting fungal growth and development. We describe the key structural elements of hydrophobins and the molecular characteristics that underlie self-assembly of these proteins at interfaces. We outline the multiple roles that hydrophobins can play in supporting aerial growth of filamentous structures, facilitating spore dispersal and preventing an immune response in the infected host. The growing understanding of the hydrophobin protein structure and self-assembly process highlights the potential for hydrophobin proteins to be engineered for use in a variety of novel applications that require biocompatible coatings.

Hydrophobins are amphiphilic proteins secreted by filamentous fungi in a soluble form, which can self-assemble at hydrophilic/hydrophobic or water/air interfaces to form amphiphilic layers that have multiple biological roles. We have investigated the conformational changes that occur upon self-assembly of six hydrophobins that form functional amyloid fibrils with a rodlet morphology. These hydrophobins are present in the cell wall of spores from different fungal species. From available structures and NMR chemical shifts, we established the secondary structures of the monomeric forms of these proteins and monitored their conformational changes upon amyloid rodlet formation or thermal transitions using synchrotron radiation circular dichroism and Fourier-transform infrared spectroscopy (FT-IR). Thermal transitions were followed by synchrotron radiation circular dichroism in quartz cells that allowed for microbubbles and hence water/air interfaces to form and showed irreversible conformations that differed from the rodlet state for most of the proteins. In contrast, thermal transitions on hermetic calcium fluoride cells showed reversible conformational changes. Heating hydrophobin solutions with a water/air interface on a silicon crystal surface in FT-IR experiments resulted in a gain in β-sheet content typical of amyloid fibrils for all except one protein. Rodlet formation was further confirmed by electron microscopy. FT-IR spectra of pre-formed hydrophobin rodlet preparations also showed a gain in β-sheet characteristic of the amyloid cross-β structure. Our results indicate that hydrophobins are capable of significant conformational plasticity and the nature of the assemblies formed by these surface-active proteins is highly dependent on the interface at which self-assembly takes place., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Rice blast is a devastating disease of rice caused by the fungus Magnaporthe oryzae and can result in loss of a third of the annual global rice harvest. Two hydrophobin proteins, MPG1 and MHP1, are highly expressed during rice blast infections. These hydrophobins have been suggested to facilitate fungal spore adhesion and to direct the action of the enzyme cutinase 2, resulting in penetration of the plant host. Therefore a mechanistic understanding of the self-assembly properties of these hydrophobins and their interaction with cutinase 2 is crucial for the development of novel antifungals. Here we report details of a study of the structure, assembly and interactions of these proteins. We demonstrate that, in vitro, MPG1 assembles spontaneously into amyloid structures while MHP1 forms a non-fibrillar film. The assembly of MPG1 only occurs at a hydrophobic:hydrophilic interface and can be modulated by MHP1 and other factors. We further show that MPG1 assemblies can much more effectively retain cutinase 2 activity on a surface after co-incubation and extensive washing compared with other protein coatings. The assembly and interactions of MPG1 and MHP1 at hydrophobic surfaces thereby provide the basis for a possible mechanism by which the fungus can develop appropriately at the infection interface.