Title

Elucidation of the Mechanism of Sc3 Hydrophobin Self-Assembly From Aqueous Media

Date of Award

2003

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Polymers and High Performance Materials

First Advisor

Charles L. McCormick

Advisor Department

Polymers and High Performance Materials

Abstract

The fungus Schizophyllum commune produces a low molecular weight (10.6 kDa) hydrophobic protein, Sc3, that is in a class of fungal proteins termed hydrophobins. All hydrophobins exhibit the unique ability to self-assemble into higher ordered complexes onto fungal surfaces, allowing for the emergence of aerial hyphae structures from aqueous media and providing protection for fungal spores. At hydrophobic/hydrophilic interfaces, the hydrophobin Sc3 self-assembles into stable films that cannot be disrupted by surfactants, organic solvents, or denaturants. We studied this interfacial self-assembly and compared it with that occurring in the absence of an interface. The latter has not been previously reported. The two types of supramolecular assemblies were examined by techniques including sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, density gradient centrifugation, phase-contrast and atomic force microscopies, and steady-state fluorescence spectroscopy. The importance of eight cysteines, conserved in all hydrophobin proteins, to the assembly process was studied by utilizing reducing agents to disrupt four intramolecular disulfide cross-links. A model, based on evidence from this study and works of others, has been proposed which defines three distinct types of Sc3 assembly: soluble U-Sc3, which is probably micellar with a few unimers held together by nonspecific hydrophobic forces; interfacially assembled I-Sc3, a large complex of highly ordered stacked β-sheets formed in a templated manner at polar/nonpolar interfaces; and a less-ordered S-Sc3 formed through time-dependent association in water in the absence of an interface. Recombinant DNA technologies were utilized to design five expression constructs for the heterologous production of Sc3 in the bacteria Echerichia coli and the yeast Pichia pastoris . Soluble, recombinant Sc3 was isolated and sequentially purified after expression in bacteria under the control of the pBAD/gIII-Sc3 construct. The purification of recombinant Sc3 from a bacterial host has never been reported. Unfortunately, the recombinant Sc3 was not functional compared with the native Sc3 from S. commune as was demonstrated by atomic force microscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The production of recombinant Sc3 was also attempted in Pichia pastoris utilizing the pPICZ and pPICZα constructs, but recombinant Sc3 was not expressed. Although unsuccessful, attempts to produce recombinant Sc3 have yielded valuable information which will likely lead to the eventual production of functional protein. If a recombinant system could be developed, site-directed mutagenesis could be utilized to modify key amino acids for the design of hydrophobins with altered properties. These altered Sc3 polypeptides could further elucidate the structure/function relationship in regards to self-assembly of I-Sc3 and S-Sc3 structures and lead to the eventual design of materials with a wide range in technical and medical applications.