Molecular Structure of Hydrophobins Studied with Site-Directed Mutagenesis and Vibrational Sum-Frequency Generation Spectroscopy

Hydrophobins are surface-active fungal proteins that adsorb to the water-air interface and self-assemble into amphiphilic, water-repelling films that have a surface elasticity that is an order of magnitude higher than other molecular films. Here we use surface-specific sum-frequency generation spectroscopy (VSFG) and site-directed mutagenesis to study the properties of class I hydrophobin (HFBI) films from Trichoderma reesei at the molecular level. We identify protein specific HFBI signals in the frequency region 1200-1700 cm-1 that have not been observed in previous VSFG studies on proteins. We find evidence that the aspartic acid residue (D30) next to the hydrophobic patch is involved in lateral intermolecular protein interactions, while the two aspartic acid residues (D40, D43) opposite to the hydrophobic patch are primarily interacting with the water solvent.