Exploring the Structural Stability and Assembly Mechanism of Hydrophobin Proteins

Hydrophobins are small, globular proteins with amphiphilic character that are produced and secreted by filamentous fungi. At hydrophobic-hydrophilic interfaces they self-assemble into durable amyloid-containing structures, called rodlets, which create protective, water repellent coatings for fungal spores. Current models of hydrophobin self-assembly predict that hydrophobin monomers undergo a conformational change at a hydrophobic-hydrophilic interface and integrate into a growing rodlet, however the mechanistic details of rodlet assembly are unknown. To investigate the assembly mechanism of hydrophobins, we carried out stability studies with SC16, a hydrophobin isolated from Schizophyllum commune. SC16 was recombinantly expressed using E. coli and purified by immobilized Ni2+ affinity chromatography. NMR spectroscopy was used to determine that the structure of SC16 was minimally perturbed by denaturing (8 M urea) or reducing (2 mM DTT) conditions. NMR-based relaxation experiments were used to identify the termini and one loop region as dynamic. The NMR data and X-ray crystal structure suggest that SC16 is a monomer in solution, which is consistent with current assembly models. Mutant forms of SC16 are being employed to determine the sequences and conformational changes required for rodlet assembly, with thioflavin T assays used to quantify amyloid formation and electron microscopy used to visualize rodlet formation. Determining which hydrophobin sequences are responsible for self-assembly will allow the rational modification of hydrophobins to add new functionalities or influence their self-assembly.