from disulfide formation at the elevated local protein concentrations present in the interfacial layer.
This is unlikely to be the origin of the observed difference in stability of WT-BslA and L77K-BslA films under compression, however, because the cysteines are unchanged. Moreover, our simulations show that the wild-type and mutant proteins are inserted into the interface in similar orientations and with the same tilt angle, suggesting that there is unlikely to be any orientational barrier to disulfide bond formation by the mutant protein at the interface.
Nonetheless, the C×C motif may have functional consequences in the biofilm, either mediating interactions between BslA molecules, or interactions with other protein or polysaccharide components in the matrix. For example, the surface layer of BslA observed in the biofilm is clearly more than a single protein layer thick (10); BslA may function as a dimeric protein with one cap region interacting with the interface and the second mediating protein–protein interactions within the biofilm. One important consequence of the mechanism we have uncovered, however, is that if the protein is indeed dimeric, it is likely to be bifunctional: one of the caps may be β-sheet and hydrophobic and the second is random coil and stable in an aqueous or hydrophilic environment. The amphiphilic nature of the fungal hydrophobins has led to suggestions for many potential applications, and these may be equally relevant to BslA. Hydrophobins have been proposed for use as surface modifiers and coating agents (30), and as emulsifiers, foam stabilizers, and surfactants in many application areas including the food industry (31). The slow kinetics of adsorption will be an important factor to consider when attempting to use BslA in any applications, particularly where other surfactants are present. It has been shown, for example, that class I hydrophobins adhere morestrongly tointerfaces thantheclass II proteins, but that the class II species can successfully compete to form a mixed interfacial membrane (32). Unlike BslA, however, class II hydrophobins exhibit no barrier to interfacial adsorption, whereas the rapid adsorption of any competing species is likely