Spectroscopic Evidence for Amyloid-like Interfacial Self-Assembly of Hydrophobin Sc3 Peter Butko,* Justin P. Buford,* ,1 J. Shawn Goodwin,* Paul A. Stroud,† Charles L. McCormick,† and Gordon C. Cannon* *Department of Chemistry & Biochemistry and †Department of Polymer Science, University of Southern Mississippi, Hattiesburg, Mississippi 39406 Received November 20, 2000 Amphipathic fungal proteins called hydrophobins are able to self-assemble into insoluble supramolecu- lar structures at hydrophobic/hydrophilic interfaces, but the molecular mechanism and underlying protein conformation changes are not known. Secondary- structure prediction indicated that hydrophobin Sc3 is an all- protein. Many amyloidogenic proteins self- assemble into insoluble amyloid fibrils while undergo- ing a change to an all- conformation. In this study we show that two dyes, thioflavin T, and Congo red, which are widely used for specific detection of stacked  sheets, interact with Sc3 assemblies in the same way as with the amyloid -sheet fibrils. We conclude that Sc3, and probably other hydrophobins too, self- assemble at interfaces in the same manner as amyloi- dogenic proteins, i.e., through -sheet stacking. © 2001 Academic Press Key Words: hydrophobin; protein self-assembly; amy- loid; -sheet stacking; thioflavin T fluorescence; Congo red. Hydrophobins are small, about 100 amino acids long, amphipathic fungal proteins that exhibit the remark- able property of self-assembling into insoluble mem- branes on hydrophobic/hydrophilic interfaces (1, 2). Be- cause of this ability, hydrophobins offer a wide range of possible applications in technology. They adhere to both hydrophilic and hydrophobic surfaces, such as glass, mica, Parafilm, and Teflon, thereby modifying their properties (2–5). Furthermore, they self-assemble on the surface of microscopic gas bubbles upon vortex- ing or gas-bubbling the protein solution (3) and on the surface of oil droplets in water (2). The latter phenom- enon has been shown to lead to efficient hydrocarbon sequestration and stabilization of aqueous oil emul- sions (6). A coherent picture of the biological significance of hydrophobins formed just recently. Hydrophobin as- semblies have been found on the surface of the fungal aerial hyphae, filamentous reproductive structures that emerge from aqueous environment into the air (7). Since hydrophobins are surface-active—they decrease the surface tension of water from 72 mN/m to 43 or 32 mN/m, values characteristic for synthetic surfactants (8, 9)—these proteins may aid the hyphae to break through the air/water interface and protect them against dehydration (10). In addition, hydrophobins may facilitate adhesion of pathogenic fungi to hydro- phobic surfaces on a host organism, such as the plant leaf or insect cuticle (2). Little is known about the hydrophobin self-assembly mechanism at the molecular level. The amino-acid se- quences of all known hydrophobins show 34% similar- ity and exhibit two common characteristic features: the presence of a short signal sequence predestining the protein for secretion and the presence of eight cysteine residues whose relative positions are conserved (1). In fact, the pattern -C-X 5–9 -C-C-X 6 –39 -C- (where C and X denote cysteine and any amino acid, respectively), present in both the N- and C-terminal halves of the molecule, is considered a signature of hydrophobins. Four disulfide bridges have been found in one hydro- phobin, cerato-ulmin: C1-C2, C3-C4, C5-C6, and C7-C8 (11). Without proof, this pattern has been tentatively assumed for all hydrophobins. The importance of the disulfide bridges for the protein self-assembly is not clear, but hydrophobin assemblies can be dissolved by trifluoroacetic acid treatment in the absence of reduc- ing agents (12) and hydrophobin is still capable of self-assembly after covalent labeling with a sulfhydryl- reactive fluorescence probe 5-((((2-iodoacetyl)amino)- ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) (Goodwin, J. S., Cannon, G. C., and Butko, P., unpub- Abbreviations used: A, amyloid  peptide (1-40); CR, Congo red; ThT, thioflavin T. 1 Participant in the University of Southern Mississippi Summer Undergraduate Research Program. Permanent address: Department of Chemistry, Delta State University, Cleveland, MS 38733. Biochemical and Biophysical Research Communications 280, 212–215 (2001) doi:10.1006/bbrc.2000.4098, available online at http://www.idealibrary.com on 212 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.