Hydrophobins are a conserved family of surface-active proteins that, among other functions, lower the surface tension of growth medium, allowing fungal hyphae to penetrate the air-water interface. The hydrophobins are divided into class I and class II; class I proteins form robust amyloid-like rodlets at the air-water interface, whereas class II proteins reduce surface tension by forming ordered lattices of native-like protein at the interface. In both classes, eight canonical cysteine residues form a highly conserved series of disulfide bridges that provide a rigid framework that restricts the mobility of the polypeptide chain (12, 13). In the class II hydrophobins, this framework is thought to stabilize the surface exposure of the large hydrophobic patch that mediates interfacial assembly (14). The presence of a large hydrophobic patch on the surface of BslA, combined with its biological function, caused us to classify BslA as a bacterial hydrophobin; however, it shares neither sequence nor structural similarity. An outstanding question remains, therefore: in the absence of a stabilizing disulfide-bonded network, how is the large surface-exposed hydrophobic patch of BslA stabilized sufficiently in aqueous environments to mediate the surface activity of the protein?
Here we use WT-BslA and a targeted mutation in the cap domain (L77K) to determine the mechanism that enables BslA to partition from the aqueous phase to the interface, where it decreases the interfacial tension and self-assembles to form an ordered rectangular 2D protein lattice. We chose the L77K mutation for further investigation as it exhibits one of the most dramatic changes in interfacial activity both in vivo and in vitro (10), thus enabling us to determine the mechanism of action. We show that BslA undergoes an environmentally responsive conformational change: the cap is stabilized in aqueous solution by