Class I hydrophobins are small fungal proteins which self-assemble into films with an underlying amyloid structure at hydrophobic:hydrophilic interfaces. The films play many roles in fungal biology including facilitating spore formation and dispersal, as well as mediating host attachment and infection. These layers adhere tightly to surfaces and reverse their wettability, making them attractive for coating and solubilizing hydrophobic materials such as medical implants and hydrophobic drugs. While a number of hydrophobin structures have been determined by us and other groups in the last decade, detailed structural and mechanistic information of the hydrophobin assembly process and the assembled films is lacking. This information would greatly assist with efforts to engineer hydrophobin-based products and the development of new anti-fungals.
We have been building a molecular picture of hydrophobin films and the assembly processes using mutagenesis, solution and solid-state NMR spectroscopy (ssNMR), solution and solid-state circular dichroism (ssCD) spectropolarimetry, mathematical modelling and molecular dynamic simulations. ssNMR experiments coupled with fast magic angle spinning have revealed a two-fold molecular composition including a substantially disordered but relatively immobile region and a structured core, with chemical shifts pointing to the presence of newly formed β-sheet structural elements. The major conformational changes upon monolayer assembly and the presence of a core structure rich in β-sheet have been further support by recent acquired ssCD spectra and 4-dimensional NOESY experiments. We are currently developing mathematical models to describe the assembly process based on these results. The implications in terms of the biology and potential applications will be discussed.