Fungal Hydrophobin Proteins Produce Self-Assembling Protein Films with Diverse Structure and Chemical Stability

Abstract

Hydrophobins are small proteins secreted by fungi and which spontaneously assemble into amphipathic layers at hydrophilic-hydrophobic interfaces. We have examined the self-assembly of the Class I hydrophobins EAS_∆15 and DewA, the Class II hydrophobin NC2 and an engineered chimeric hydrophobin. These Class I hydrophobins form layers composed of laterally associated fibrils with an underlying amyloid structure. These two Class I hydrophobins, despite showing significant conformational differences in solution, self-assemble to form fibrillar layers with very similar structures and require a hydrophilic-hydrophobic interface to trigger self-assembly. Addition of additives that influence surface tension can be used to manipulate the fine structure of the protein films. The Class II hydrophobin NC2 forms a mesh-like protein network and the engineered chimeric hydrophobin displays two multimeric forms, depending on assembly conditions. When formed on a graphite surface, the fibrillar EAS_∆15 layers are resistant to alcohol, acid and basic washes. In contrast, the NC2 Class II monolayers are dissociated by alcohol treatment but are relatively stable towards acid and base washes. The engineered chimeric Class I/II hydrophobin shows increased stability towards alcohol and acid and base washes. Self-assembled hydrophobin films may have extensive applications in biotechnology where biocompatible; amphipathic coatings facilitate the functionalization of nanomaterials.

Keywords: amphipathic; film; hydrophobin; protein; self-assembly.

Figure 1

Solution structures of the soluble forms of Class I hydrophobins: (a) EAS_∆15 and (b) DewA, determined by nuclear magnetic resonance (NMR) spectroscopy. Ribbon representations of protein molecular structure were prepared from Protein Data Bank (PDB) entries 2K6A and 2LSH using the molecular graphics program PyMol [24]. (c) Rodlet assembly by EAS_∆15 and DewA assayed by Thioflavin T (ThT) fluorescence demonstrates that these Class I hydrophobins only assemble into amyloid-like rodlets after introduction of a hydrophilic-hydrophobic interface. Transmission electron microscopy (TEM) of negatively stained: (d) EAS_∆15 and (e) DewA hydrophobin rodlet monolayers, transferred from the surface of protein droplets and imaged through the holes in a holey film. (f) Atomic force microscopy (AFM) of the rodlets formed when a 5 µg/mL DewA solution was allowed to dry overnight onto a highly oriented pyrolytic graphite (HOPG) surface.

Figure 2

TEM image of negatively stained DewA and EAS_∆15 hydrophobin rodlet-containing layers transferred from the surface of droplets of solutions containing: (a) DewA 15% (v/v) ethanol; (b) EAS_∆15 15% (v/v) ethanol; (c) DewA 25% (v/v) ethanol; and (d) EAS_∆15 25% (v/v) ethanol after incubation at room temperature for 20 min.

Figure 3

(a) AFM topographic scan of HOPG surface coated with a layer of EAS_∆15 protein after a 50-µL drop of EAS_∆15 (25 µg/mL) was incubated for 1 min and transferred onto a freshly cleaved HOPG surface; multiple protein rafts are observed; (b) AFM scan of HOPG surface coated with a layer of EAS_∆15 protein after a 50 µL drop of EAS_∆15 (5 µg/mL) was left to dry overnight onto a freshly cleaved HOPG surface. Sample was imaged directly after drying; (c) a 1-min wash to remove loosely bounded protein layers reveals an underlying, ordered single layer rodlet film; and (d) after 7 min of washing with a stream of running Milli-Q^® water (MQW), loose fibril layers are removed and a highly ordered layer of rodlets remains attached to the HOPG.

Figure 4

AFM scans of EAS_∆15 rodlets on HOPG treated for 5 min with different solvents, followed by a 2-min water wash: (a) exposure to up to 100% ethanol does not affect the structure of the EAS_∆15 rodlet layer and effectively cleans the surface of any loosely bound protein; (b) EAS_∆15 fibrillar film is disrupted by treatment with 100% trifluoroacetic acid (TFA); fibrillar rodlet structure is stable towards treatment with: (c) 3M NaOH and (d) 3M HCl, but residual impurities remain attached to the fibrils and are not easily removed by washing.

Figure 5

(a) Ribbon representation of the solution structure of NC2, prepared from PDB Entry 4AOG using PyMol [24]; (b) NC2 layer washed with MQW for 5 min, displaying a protein network with pores of 20–30 nm and a layer height of 1.5–2 nm; (c) NC2 layer after treatment with 60% ethanol for 5 min; (d) NC2 layer after treatment with 3 M NaOH; and (e) NC2 layer after treatment with 3 M HCl.

Figure 6

(a) Sequence alignment illustrating the construction of the chimeric hydrophobin NChi2 from the proteins NC2 and EAS (named for its easily wettable spore phenotype). The Cys7–Cys8 region of NC2 is shorter than the corresponding region in EAS and EAS_∆15, this difference is indicated with a dotted line. The 15-residue deletion from EAS, to generate EAS_∆15, is indicated by a solid line. The conservation of the amyloidogenic region between EAS, EAS_∆15 and NChi2 is indicated in red; (b) TEM image of negatively stained NChi2 rodlets formed under conditions of low pH (=2.5) and elevated temperature (45 °C) with extended shaking. (c) AFM image of NChi2 dried down from pH = 2.5 at 45 °C and then washed with MQW for 5 min.

Figure 7

Morphology and stability of NChi2 layers formed from protein at under native conditions: (a) NChi2 coating washed with MQW has Class II morphology; (b) treatment with 60% ethanol does not disrupt the layer; (c) the protein network attached to HOPG is stable towards 3M NaOH; and (d) treatment with 3M HCl disrupts interactions between protein molecules, loosening the mesh structure.

Figure 8

Schematic representation of the monomeric and self-assembled forms of Class I, Class II and chimeric hydrophobins. Grafting of the fibril-forming segment from a Class I hydrophobin onto a Class II hydrophobin can confer additional self-assembly character, as evidenced by the chimera NChi2 which is able to form both a Class II-like polygonal protein network or Class I-like fibrillar rodlet structures depending on assembly conditions.