(84v) A Structure-Guided Design of an Oligomeric Hydrophobin Bundle Using Coiled Coils

The class II hydrophobins, HFBI, are amphiphilic proteins secreted by filamentous fungi called Trichoderma reesei. These proteins are amphiphilic with a signature hydrophobic patch and can self-assemble at air-water or oil-water interfaces, forming highly ordered membrane-like structures. Due to their unique amphiphilicity property, hydrophobins have been utilized in various applications, such as emulsification, drug solubilization, protein purification, and functional materials fabrication. However, genetic modifications are restricted in their native fungal host, T. reesei. In contrast, the heterologous expression from yeast or bacterial hosts provides the benefits such as inexpensive nutrients requirement, rapid accumulation, and simpler industrial scale-up. However, forming the correct disulfide bonds buried in hydrophobins is challenging in bacterial expression systems. In addition, genetic modifications of hydrophobins for functionalization, without compromising their self-assembling property, are desirable but with limited success. We hypothesize that the genetic fusion of HFBI with an oligomeric protein motif whose shape matches the lattice geometry of self-assembled HFBI would direct the correct folding and protein self-assembly, improve protein expression, and enable functionalization. In this study, we designed fusion proteins of HFBI and a hexameric helical protein (HexCC), demonstrated their expression and purification from a bacterial host system, and characterized their interfacial assembly for functional coating materials fabrication.

We designed the recombinant fusion protein, HFBI-HexCC, by aligning HexCC with the hexagonal lattice of HFBI along their axes of symmetry. HexCC is a coiled-coil protein that forms a bundle-like hexameric oligomer. The sequences of HFBI and HexCC were fused using various unstructured linker sequences, followed by building comparative structure models from the crystal structure templates. From the results, we chose a linker sequence that showed the best geometric alignment without disrupting the overall structures of HFBI-HexCC. We performed the normal mode analysis to investigate the protein conformation and flexibility dynamics and confirmed the structural alignment between HFBI and HexCC. We also designed a fusion protein variant, HFBI_MC-HexCC, where the eight cysteine residues were mutated to alanine or valine to investigate the effects of coiled-coil fusion on the disulfide bond formation and protein folding. After the transformation of Escherichia coli cells with DNA plasmids, the fusion proteins were expressed, purified, and characterized. Both proteins were soluble after expression from E. coli, and the major fraction of proteins was in the form of hexamers or larger complexes. The fusion proteins were capable of self-assembling on the surface of polytetrafluoroethylene (PTFE) films, which was confirmed by the reduction in the surface hydrophobicity. Due to the protein layer formation on the surface PTFE, the water contact angle decreased from ~106° to ~73°. The mutants HFBI_MC-HexCC also showed a similar property. We also tested protein self-assembly on a polystyrene surface and investigated their potential as anti-fouling coating materials. Polystyrene microwell plates were coated with both proteins, and the adherence of E. coli cells was studied by quantifying the fluorescence of the green fluorescent protein, which is expressed inside the cells. The protein-coated polystyrene surfaces showed a twenty-fold reduction in the number of adhered cells compared to the bare control surfaces.

In conclusion, we demonstrated the design of oligomeric amphiphilic fusion proteins derived from fungal hydrophobin. Guided by the computational structural modeling, we fused HFBI with a coiled-coil, which resulted in a successful protein expression in a bacterial host system. In addition, the fusion proteins exhibited oligomer formation and self-assembling properties on polymer surfaces. These fusion proteins can be used for biocompatible modification of various hydrophobic materials. Functionalization by further genetic modifications is possible, enabling them to serve as surface-binding agents with promising potential in biomedical, surface adhesion, and coatings applications.