Solution structure and interface‐driven self‐assembly of NC2, a new member of the Class II hydrophobin proteins

Abstract

Hydrophobins are fungal proteins that self-assemble spontaneously to form amphipathic monolayers at hydrophobic:hydrophilic interfaces. Hydrophobin assemblies facilitate fungal transitions between wet and dry environments and interactions with plant and animal hosts. NC2 is a previously uncharacterized hydrophobin from Neurospora crassa. It is a highly surface active protein and is able to form protein layers on a water:air interface that stabilize air bubbles. On a hydrophobic substrate, NC2 forms layers consisting of an ordered network of protein molecules, which dramatically decrease the water contact angle. The solution structure and dynamics of NC2 have been determined using nuclear magnetic resonance spectroscopy. The structure of this protein displays the same core fold as observed in other hydrophobin structures determined to date, including the Class II hydrophobins HFBI and HFBII from Trichoderma reesei, but certain features illuminate the structural differences between Classes I and II hydrophobins and also highlight the variations between structures of Class II hydrophobin family members. The unique properties of hydrophobins have attracted much attention for biotechnology applications. The insights obtained through determining the structure, biophysical properties and assembly characteristics of NC2 will facilitate the development of hydrophobin-based applications.

Applications of hydrophobins: current state and perspectives

“…3). The class II hydrophobin NC2 is predominantly present in the monomeric form up to concentrations of 5 mg mL −1 (Ren et al 2013b). HFBI and HFBII only form monomers at very low concentrations (1-2 μg mL −1…”

“…The 3D structure of the soluble state of the class I hydrophobins EAS (Neurospora crassa), DewA (Aspergillus nidulans), MPG1 (Magnaporthe grisea), and RodA (Aspergillus fumigatus) (Kwan et al 2006;Morris et al 2012Morris et al , 2013Rey et al 2013;Pille et al 2014) and the class II hydrophobins HFBI, HFBII (Trichoderma reesei), and NC2 (N. crassa) (Hakanpää et al 2004(Hakanpää et al , 2006aRen et al 2013b) has been solved. Both types of hydrophobin contain a four-stranded β-barrel core (Fig.…”

“…In contrast, the Nterminal sequence (not shown) of DewA, RodA, and MPG1 and loops L1 and L2 contain α-helical structure (Fig. 2b) (Ren et al 2013b;Pille et al 2014). Charged residues of class I and class II hydrophobins (EAS, DewA, and NC2) were shown to be localized at one side of the surface of the protein, leaving the other side without charge (Kwan et al 2006;Morris et al 2013;Ren et al 2013b).…”

“…2b) (Ren et al 2013b;Pille et al 2014). Charged residues of class I and class II hydrophobins (EAS, DewA, and NC2) were shown to be localized at one side of the surface of the protein, leaving the other side without charge (Kwan et al 2006;Morris et al 2013;Ren et al 2013b). At this side, discrete hydrophobic patches are present in DewA, NC2, EAS, HFBI, and HFBII (ranging in size between 424 and 891 Å 2 ) giving these molecules an amphipathic nature.…”

Characterization of a Basidiomycota hydrophobin reveals the structural basis for a high-similarity Class I subdivision

“…While hydrophobins from both phyla have been characterized, high-resolution structures exist only for those of ascomycota origin: the Class I EAS, DewA, MPG1, and Class II NC2, HFBI, and HFBII81920212223. To address whether basidiomycota hydrophobins share structural and functional features with these proteins, we preliminarily characterized SC16 ( hyd1 ) from Schizophyllum commune and determined its structure.…”