Filling in the gaps of the spectrum-wide characterization of hydrophobins: An interface-active protein family with industrial potential

Hydrophobins are secreted non-catalytic fungal proteins that act at interfaces whose functions range from self-assembling to form a fungal â raincoatâ on the fruiting body, to recruiting enzyme activities to surfaces. Traditionally subdivided into two classes (I and II) based on primary sequence and observed solution characteristics, the underlying thesis of this work is that classification of type I hydrophobins can be further refined. As such, the analysis of the predicted primary sequences of hydrophobins originating from the genomes of over 200 fungal species was conducted by sequence-alignment principal component analysis (SA-PCA) revealing a high-identity class I Basidiomycota subdivision. To experimentally validate this subdivision, the structure of one of its members, the predicted Schizophyllum commune hydrophobin SC16 (hyd1), was solved by solution-NMR. While sharing the core hydrophobin structure, structural elements believed to be necessary for class I behaviour were absent. However, the protein was an active class I hydrophobin as demonstrated by specific binding to the amyloid-detecting dye Thioflavin T and rodlet formation at the air-water interface visualized by atomic force microscopy (AFM). Together, these results reveal a structural basis for the class IB subdivision. Next, methods to further characterize the spectrum of hydrophobin sequences were explored. Pichia pastoris was found to be an effective host for the soluble recombinant expression of the three predicted hydrophobins of the Ascomycota entomopathogen Cordyceps militaris genome. Of these solubly expressed proteins, the preliminary visualization of class I type rodlets by AFM of one of the three (CMil3) is discussed. In the final chapter the matrix-free chromatographic technique of flow field-flow fractionation was harnessed to study the oligomeric solution-state of hydrophobins. This dissertation concludes with next steps in applying the framework used for the sequence analyses as a means of mapping and testing the bounds of the unique properties exhibited by this protein family.Ph.D