Experimental Evidence for Multiple Assembled States of Sc3 from Schizophyllum commune Paul A. Stroud, † J. Shawn Goodwin, ‡ Peter Butko, ‡ Gordon C. Cannon, ‡ and Charles L. McCormick* ,†,‡ Department of Polymer Science and Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, Mississippi 39406-0076 Received February 11, 2003; Revised Manuscript Received April 25, 2003 The hydrophobin Sc3 from the fungus Schizophyllum commune assembles from the aqueous phase into ordered structures with substantially different characteristics depending upon experimental conditions. Under the first condition, a vortexing procedure widely reported in the literature, interfacial assembly yields highly ordered, stacked -sheets. We have also observed a previously unreported assembly of Sc3 under a second condition, which occurs in a time-dependent manner from quiescent solution. The resulting types of assembled states have been compared utilizing fluorescence techniques, sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, density gradient centrifugation, and phase contrast and atomic force microscopy. A model based on this study and previous literature is proposed that suggests three distinct states of Sc3: (1) soluble Sc3 consisting of unimers or multimers in micelle-like association, (2) interfacially assembled I-Sc3 with highly ordered, stacked -sheets, presumably formed in a templated manner at the air/water interface of microscopic bubbles generated by vortexing, and (3) solution-assembled S-Sc3, a less-ordered structure formed in a time-dependent manner in the absence of an interface. Introduction Hydrophobins are a group of amphipathic fungal proteins possessing remarkable structural characteristics that allow for unique self-assembling behavior. These characteristics have spawned a flurry of research activity including that focused on pharmaceutical and other medical applications. 1-5 Of primary interest is the ability of hydrophobins dissolved in aqueous media to readily organize into insoluble amphi- pathic films at hydrophobic/hydrophilic interfaces. Hydro- phobins are low molecular weight (8-10 kDa) polypeptides that contain eight conserved cysteines in their sequence. The conserved cysteines are intramolecularly cross-linked and are considered important for maintaining protein solubility and avoiding premature self-association. 1 Hydrophobins can be characterized as either class I or class II on the basis of their solubility characteristics and hydropathy patterns, 2-4 yet both classes show a similar propensity to self-assemble into approximately 10 nm thick amphipathic insoluble membranes. 5 Class I films are not disrupted when treated with surfactants, solvents, and denaturing agents (e.g., urea). 4,6 Only trifluoroacetic acid (TFA) has been found to dissociate these supramolecular structures. 6 By contrast, class II films are easily disrupted with surfactants or solvent mixtures such as 60% ethanol/ water. 7 Though many class I and class II hydrophobins have been identified and isolated, the most well-studied hydro- phobin to date is Sc3, a class I hydrophobin from the fungus Schizophyllum commune. 4-6,8-11 When air/water interfaces are created in hydrophobin solutions (i.e., by bubbling gas through the solution), protein- coated air bubbles result that can be visualized by light microscopy. 6,7 Hydrophobins also stabilize oil droplets in solution. 11,12 For example, Sc3 and oil mixtures extruded through membranes of fixed pore sizes result in uniform protein-coated vesicles. 12 In addition to the stabilization of bubbles and oil droplets, hydrophobins can modify both hydrophobic and hydrophilic surfaces as applied from aqueous solution. Preferential surface orientation arises from the amphipathic nature of Sc3. 10-14 Amino acid sequences of hydrophobins contain distinct hydrophilic and hydrophobic regions, and it has been proposed that hydrophobins may resemble a polymeric surfactant with high surface activity. 10,11,15-17 The hydropathy plot of Sc3 indicates that approximately the first third of the amino acid sequence from the N terminus is relatively hydrophilic compared to the remaining portion. 2,3 In addition, Sc3 contains approximately 20 mannose residues, thought to be O-linked through threonines in the N-terminal third of the protein, further increasing the hydrophilicity of that region of the polypeptide chain. 10 The Sc3 protein in its unimeric or “unassociated” form is comprised of a high proportion of -sheet structure (41%), as determined by circular dichroism (CD) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. 4,5,10 When Sc3 is induced to self-organize at the air/water or Teflon/water interface, a small, transient increase in R-helical secondary structure is observed, followed by an increase in -sheet structure. The * To whom correspondence should be addressed. E-mail: Charles. McCormick@usm.edu. † Department of Polymer Science. ‡ Department of Chemistry and Biochemistry. 956 Biomacromolecules 2003, 4, 956-967 10.1021/bm034045e CCC: $25.00 © 2003 American Chemical Society Published on Web 06/05/2003