Structural and Functional Role of the Disulfide Bridges in the Hydrophobin SC3

Abstract

Hydrophobins function in fungal development by self-assembly at hydrophobic-hydrophilic interfaces such as the interface between the fungal cell wall and the air or a hydrophobic solid. These proteins contain eight conserved cysteine residues that form four disulfide bonds. To study the effect of the disulfide bridges on the self-assembly, the disulfides of the SC3 hydrophobin were reduced with 1,4-dithiothreitol. The free thiols were then blocked with either iodoacetic acid (IAA) or iodoacetamide (IAM), introducing eight or zero negative charges, respectively. Circular dichroism and infrared spectroscopy showed that after opening of the disulfide bridges SC3 is initially unfolded. IAA-SC3 did not self-assemble at the air-water interface upon shaking an aqueous solution. Remarkably, after drying down IAA-SC3 or after exposing it to Teflon, it refolded into a structure similar to that observed for native SC3 at these interfaces. Iodoacetamide-SC3 on the other hand, which does not contain extra charges, spontaneously refolded in water in the amyloid-like β-sheet conformation, characteristic for SC3 assembled at the water-air interface. From this we conclude that the disulfide bridges of SC3 are not directly involved in self-assembly but keep hydrophobin monomers soluble in the fungal cell or its aqueous environment, preventing premature self-assembly. Hydrophobins function in fungal development by self-assembly at hydrophobic-hydrophilic interfaces such as the interface between the fungal cell wall and the air or a hydrophobic solid. These proteins contain eight conserved cysteine residues that form four disulfide bonds. To study the effect of the disulfide bridges on the self-assembly, the disulfides of the SC3 hydrophobin were reduced with 1,4-dithiothreitol. The free thiols were then blocked with either iodoacetic acid (IAA) or iodoacetamide (IAM), introducing eight or zero negative charges, respectively. Circular dichroism and infrared spectroscopy showed that after opening of the disulfide bridges SC3 is initially unfolded. IAA-SC3 did not self-assemble at the air-water interface upon shaking an aqueous solution. Remarkably, after drying down IAA-SC3 or after exposing it to Teflon, it refolded into a structure similar to that observed for native SC3 at these interfaces. Iodoacetamide-SC3 on the other hand, which does not contain extra charges, spontaneously refolded in water in the amyloid-like β-sheet conformation, characteristic for SC3 assembled at the water-air interface. From this we conclude that the disulfide bridges of SC3 are not directly involved in self-assembly but keep hydrophobin monomers soluble in the fungal cell or its aqueous environment, preventing premature self-assembly. 1,4-dithiothreitol iodoacetic acid iodoacetamide thioflavin T Hydrophobins are small, moderately hydrophobic proteins characterized by similar hydropathy patterns and the presence of eight cysteine residues with conserved spacing. They fulfill a broad spectrum of functions in fungal development (1Wessels J.G.H. Adv. Microb. Physiol. 1997; 38: 1-45Crossref PubMed Google Scholar). Hydrophobins are secreted by filamentous fungi into their aqueous environment, where they self-assemble at the interface between the medium and the air. This results in a dramatic drop of the water surface tension, enabling fungi to breach the interface to form aerial reproductive structures (2Wösten H.A.B. van Wetter M.-A. Lugones L.G. van der Mei H.C. Busscher H.J. Wessels J.G.H. Curr. Biol. 1999; 9: 85-88Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Hydrophobins also provide these aerial structures with a hydrophobic coating (1Wessels J.G.H. Adv. Microb. Physiol. 1997; 38: 1-45Crossref PubMed Google Scholar) and mediate attachment of fungi to hydrophobic surfaces (for example, in pathogenic interactions) (3Wösten H.A.B. Schuren F.H.J. Wessels J.G.H. EMBO J. 1994; 13: 5848-5854Crossref PubMed Scopus (290) Google Scholar, 4Talbot N.J. Kershaw M. Wakley G.E. de Vries O.M.H. Wessels J.G.H. Hamer J.E. Plant Cell. 1996; 8: 985-999Crossref PubMed Scopus (295) Google Scholar).SC3 of Schizophyllum commune is the best studied hydrophobin, but other hydrophobins show similar properties. SC3 is a glycoprotein of 112 amino acids containing 16–22 mannose residues that are probably linked to 12 threonines and 1 serine at the amino-terminal part of the molecule (5de Vocht M.L. Scholtmeijer K. van der Vegte E.W. de Vries O.M.H. Sonveaux N. Wösten H.A.B. Ruysschaert J.M. Hadziioannou G. Wessels J.G.H. Robillard G.T. Biophys. J. 1998; 74: 2059-2068Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). SC3 is a remarkable protein because it forms an insoluble amphipathic protein film after self-assembly at a hydrophobic-hydrophilic interface that cannot be dissociated by heating in 2% SDS at 100 °C (6Wösten H.A.B. de Vries O.M.H. Wessels J.G.H. Plant Cell. 1993; 5: 1567-1574Crossref PubMed Scopus (253) Google Scholar). The hydrophobic side of this protein film is characterized by a mosaic of parallel rodlets (6Wösten H.A.B. de Vries O.M.H. Wessels J.G.H. Plant Cell. 1993; 5: 1567-1574Crossref PubMed Scopus (253) Google Scholar) of an amyloid-like nature. 1M. L. de Vocht, I. Reviakine, H. A. B. Wösten, A. Brisson, J. G. H. Wessels, and G. T. Robillard, manuscript in preparation.1M. L. de Vocht, I. Reviakine, H. A. B. Wösten, A. Brisson, J. G. H. Wessels, and G. T. Robillard, manuscript in preparation. Upon self-assembly, the different conformations of SC3 are observed depending on the nature of the interface. For instance, after binding to a hydrophobic solid the protein is trapped in a so-called “α-helical state” (5de Vocht M.L. Scholtmeijer K. van der Vegte E.W. de Vries O.M.H. Sonveaux N. Wösten H.A.B. Ruysschaert J.M. Hadziioannou G. Wessels J.G.H. Robillard G.T. Biophys. J. 1998; 74: 2059-2068Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). With the proper treatment this state can be converted to the β-sheet state, 2M. L. de Vocht, I. Reviakine, H. A. B. Wösten, A. Brisson, J. G. H. Wessels, and G. T. Robillard, manuscript in preparation.2M. L. de Vocht, I. Reviakine, H. A. B. Wösten, A. Brisson, J. G. H. Wessels, and G. T. Robillard, manuscript in preparation. which is also found after assembly on the air-water interface (5de Vocht M.L. Scholtmeijer K. van der Vegte E.W. de Vries O.M.H. Sonveaux N. Wösten H.A.B. Ruysschaert J.M. Hadziioannou G. Wessels J.G.H. Robillard G.T. Biophys. J. 1998; 74: 2059-2068Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). All hydrophobins studied so far have the same spatial distribution of cysteine residues, which is as follows:X 2–38-C-X 5–9-C-C-X 11–44-C-X 8–23-C-X 5–9-C-C-X 6–18-C-X 2–14.The initial idea that intermolecular disulfide bridges were formed during the self-assembly and thus stabilize the protein film was disproved when it was shown that SC3 self-assembled not only on air bubbles but also on non-oxidative nitrogen gas bubbles (6Wösten H.A.B. de Vries O.M.H. Wessels J.G.H. Plant Cell. 1993; 5: 1567-1574Crossref PubMed Scopus (253) Google Scholar). Moreover, it was shown that monomeric SC3 contained no free cysteine residues and that the protein film could be disassembled by treatment with trifluoroacetic acid in the absence of reducing agent (7de Vries O.M.H. Fekkes M.P. Wösten H.A.B. Wessels J.G.H. Arch. Microbiol. 1993; 159: 330-335Crossref Scopus (176) Google Scholar).SC3 is a so-called class I hydrophobin (1Wessels J.G.H. Adv. Microb. Physiol. 1997; 38: 1-45Crossref PubMed Google Scholar). The arrangement of the disulfide bridges has been determined for the class II hydrophobin cerato-ulmin (8Yaguchi M. Pusztai-Carey M. Roy C. Surewicz W.K. Carey P.R. Stevenson K.J. Richards W.C. Takai S. Sticklen M.D. Sherald J.L. Dutch Elm Disease Research: Cellular and Molecular Approaches. Springer-Verlag New York Inc., New York1993: 152-170Crossref Google Scholar). Cys1 is bonded to Cys2or Cys3; Cys2 or Cys3 is bonded to Cys4; Cys5 is bonded to Cys6 or Cys7; and Cys6 or Cys7 is bonded to Cys8. This results in a primary structure with four loops and two domains. Although there is no direct proof, the general assumption is that a similar pattern of disulfide bridges exists in SC3 (Fig. 1).Disulfide bonds can fulfill diverse functions in proteins. In general they stabilize proteins, but the exact manner by which the stabilizing effect is achieved cannot be predicted adequately by current theories (9Betz S.F. Protein Sci. 1993; 2: 1551-1558Crossref PubMed Scopus (368) Google Scholar). The conserved pattern of the cysteine residues in all hydrophobins suggests that the cysteine residues are important for hydrophobin structure and/or function. To study the role of the disulfide bridges in SC3, all disulfides were reduced with DTT,3 and the free cysteines were blocked with iodoacetic acid or iodoacetamide. The secondary structure of carboxymethylated SC3 before and after the self-assembly processes was determined. The strength of binding to a hydrophobic Teflon surface and the ability to self-assemble in the amyloid-like form and to form rodlets were also monitored.DISCUSSIONSC3 is a very stable protein. The CD spectrum of the monomeric form hardly changes in 10 mm DTT or in 5.4 mguanidine (Fig. 2). In addition, heating to 90 °C or changing the pH between 3 and 12 does not change the secondary structure of the protein (data not shown). SC3 only unfolds after reduction of the disulfide bridges with 10 mm DTT in 5.4 m guanidine. This shows that the disulfide bridges are essential for the stability of the monomeric form of SC3.In general the conformation of proteins that adsorb on a hydrophobic solid support varies from a native or nearly native conformation to an extensively unfolded conformation (18Brash J.L. Horbett T.A. Horbett T.A. Brash J.L. Protein at Interfaces II: Fundamentals and Applications. 602. American Chemical Society, Washington, D. C.1995: 1-23Google Scholar, 19Haynes C.A. Norde W. J. Colloid Interface Sci. 1995; 169: 313-328Crossref Scopus (352) Google Scholar). The remarkable refolding of IAA-SC3 on Teflon, with eight additional negative charges, demonstrates the high propensity of the hydrophobin to refold on a hydrophobic surface and is a clear illustration of the specific interaction of hydrophobins with hydrophobic substrates. However, the stability of the IAA-SC3 layer is decreased compared with a layer of assembled native SC3. This instability is not caused by a difference in structure but by the negative charges at pH 7.0, as shown by the recovery of the strong binding when the extraction with SDS was performed at low pH. Furthermore, the IAA-SC3 is still capable of forming rodlets (Fig.7 A). This confirms the observation that intermolecular disulfide bridges are not involved in the self-assembly process.All hydrophobins have the same distribution of cysteine residues, and they probably have the same pattern of disulfide bridges. They are produced as monomeric proteins and can self-assemble into more or less insoluble films after interaction with a hydrophobic-hydrophilic interface. For SC3 it has been shown that this film consists of amyloid fibrils1. For many amyloid proteins the polymerization is initiated by a partially folded state of the protein (20Taubes G. Science. 1996; 271: 1493-1495Crossref PubMed Scopus (131) Google Scholar, 21Kelly J.W. Curr. Opin. Struct. Biol. 1998; 8: 101-106Crossref PubMed Scopus (947) Google Scholar). Therefore it is understandable that opening of the disulfide bridges (which destabilizes the monomeric form) increases the propensity of hydrophobins to form amyloid fibrils.IAM-SC3 in water spontaneously self-assembles in an amyloid-like β-sheet form in the absence of a hydrophobic-hydrophilic interface, but it first goes through a short-lived α-helical state. This corroborates the observation that the α-helical form also in native SC3 is an intermediate in the self-assembly process2. We propose that the disulfide bridges stabilize the monomeric form of hydrophobins and prevent premature self-assembly (before interaction with a hydrophobic-hydrophilic interface), which would result in protein aggregates within the cell or its aqueous environment. The eight conserved cysteine residues in hydrophobins are thus essential for the function of hydrophobins at hydrophobic-hydrophilic interfaces during fungal development. Hydrophobins are small, moderately hydrophobic proteins characterized by similar hydropathy patterns and the presence of eight cysteine residues with conserved spacing. They fulfill a broad spectrum of functions in fungal development (1Wessels J.G.H. Adv. Microb. Physiol. 1997; 38: 1-45Crossref PubMed Google Scholar). Hydrophobins are secreted by filamentous fungi into their aqueous environment, where they self-assemble at the interface between the medium and the air. This results in a dramatic drop of the water surface tension, enabling fungi to breach the interface to form aerial reproductive structures (2Wösten H.A.B. van Wetter M.-A. Lugones L.G. van der Mei H.C. Busscher H.J. Wessels J.G.H. Curr. Biol. 1999; 9: 85-88Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Hydrophobins also provide these aerial structures with a hydrophobic coating (1Wessels J.G.H. Adv. Microb. Physiol. 1997; 38: 1-45Crossref PubMed Google Scholar) and mediate attachment of fungi to hydrophobic surfaces (for example, in pathogenic interactions) (3Wösten H.A.B. Schuren F.H.J. Wessels J.G.H. EMBO J. 1994; 13: 5848-5854Crossref PubMed Scopus (290) Google Scholar, 4Talbot N.J. Kershaw M. Wakley G.E. de Vries O.M.H. Wessels J.G.H. Hamer J.E. Plant Cell. 1996; 8: 985-999Crossref PubMed Scopus (295) Google Scholar). SC3 of Schizophyllum commune is the best studied hydrophobin, but other hydrophobins show similar properties. SC3 is a glycoprotein of 112 amino acids containing 16–22 mannose residues that are probably linked to 12 threonines and 1 serine at the amino-terminal part of the molecule (5de Vocht M.L. Scholtmeijer K. van der Vegte E.W. de Vries O.M.H. Sonveaux N. Wösten H.A.B. Ruysschaert J.M. Hadziioannou G. Wessels J.G.H. Robillard G.T. Biophys. J. 1998; 74: 2059-2068Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). SC3 is a remarkable protein because it forms an insoluble amphipathic protein film after self-assembly at a hydrophobic-hydrophilic interface that cannot be dissociated by heating in 2% SDS at 100 °C (6Wösten H.A.B. de Vries O.M.H. Wessels J.G.H. Plant Cell. 1993; 5: 1567-1574Crossref PubMed Scopus (253) Google Scholar). The hydrophobic side of this protein film is characterized by a mosaic of parallel rodlets (6Wösten H.A.B. de Vries O.M.H. Wessels J.G.H. Plant Cell. 1993; 5: 1567-1574Crossref PubMed Scopus (253) Google Scholar) of an amyloid-like nature. 1M. L. de Vocht, I. Reviakine, H. A. B. Wösten, A. Brisson, J. G. H. Wessels, and G. T. Robillard, manuscript in preparation.1M. L. de Vocht, I. Reviakine, H. A. B. Wösten, A. Brisson, J. G. H. Wessels, and G. T. Robillard, manuscript in preparation. Upon self-assembly, the different conformations of SC3 are observed depending on the nature of the interface. For instance, after binding to a hydrophobic solid the protein is trapped in a so-called “α-helical state” (5de Vocht M.L. Scholtmeijer K. van der Vegte E.W. de Vries O.M.H. Sonveaux N. Wösten H.A.B. Ruysschaert J.M. Hadziioannou G. Wessels J.G.H. Robillard G.T. Biophys. J. 1998; 74: 2059-2068Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). With the proper treatment this state can be converted to the β-sheet state, 2M. L. de Vocht, I. Reviakine, H. A. B. Wösten, A. Brisson, J. G. H. Wessels, and G. T. Robillard, manuscript in preparation.2M. L. de Vocht, I. Reviakine, H. A. B. Wösten, A. Brisson, J. G. H. Wessels, and G. T. Robillard, manuscript in preparation. which is also found after assembly on the air-water interface (5de Vocht M.L. Scholtmeijer K. van der Vegte E.W. de Vries O.M.H. Sonveaux N. Wösten H.A.B. Ruysschaert J.M. Hadziioannou G. Wessels J.G.H. Robillard G.T. Biophys. J. 1998; 74: 2059-2068Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). All hydrophobins studied so far have the same spatial distribution of cysteine residues, which is as follows:X 2–38-C-X 5–9-C-C-X 11–44-C-X 8–23-C-X 5–9-C-C-X 6–18-C-X 2–14. The initial idea that intermolecular disulfide bridges were formed during the self-assembly and thus stabilize the protein film was disproved when it was shown that SC3 self-assembled not only on air bubbles but also on non-oxidative nitrogen gas bubbles (6Wösten H.A.B. de Vries O.M.H. Wessels J.G.H. Plant Cell. 1993; 5: 1567-1574Crossref PubMed Scopus (253) Google Scholar). Moreover, it was shown that monomeric SC3 contained no free cysteine residues and that the protein film could be disassembled by treatment with trifluoroacetic acid in the absence of reducing agent (7de Vries O.M.H. Fekkes M.P. Wösten H.A.B. Wessels J.G.H. Arch. Microbiol. 1993; 159: 330-335Crossref Scopus (176) Google Scholar). SC3 is a so-called class I hydrophobin (1Wessels J.G.H. Adv. Microb. Physiol. 1997; 38: 1-45Crossref PubMed Google Scholar). The arrangement of the disulfide bridges has been determined for the class II hydrophobin cerato-ulmin (8Yaguchi M. Pusztai-Carey M. Roy C. Surewicz W.K. Carey P.R. Stevenson K.J. Richards W.C. Takai S. Sticklen M.D. Sherald J.L. Dutch Elm Disease Research: Cellular and Molecular Approaches. Springer-Verlag New York Inc., New York1993: 152-170Crossref Google Scholar). Cys1 is bonded to Cys2or Cys3; Cys2 or Cys3 is bonded to Cys4; Cys5 is bonded to Cys6 or Cys7; and Cys6 or Cys7 is bonded to Cys8. This results in a primary structure with four loops and two domains. Although there is no direct proof, the general assumption is that a similar pattern of disulfide bridges exists in SC3 (Fig. 1). Disulfide bonds can fulfill diverse functions in proteins. In general they stabilize proteins, but the exact manner by which the stabilizing effect is achieved cannot be predicted adequately by current theories (9Betz S.F. Protein Sci. 1993; 2: 1551-1558Crossref PubMed Scopus (368) Google Scholar). The conserved pattern of the cysteine residues in all hydrophobins suggests that the cysteine residues are important for hydrophobin structure and/or function. To study the role of the disulfide bridges in SC3, all disulfides were reduced with DTT,3 and the free cysteines were blocked with iodoacetic acid or iodoacetamide. The secondary structure of carboxymethylated SC3 before and after the self-assembly processes was determined. The strength of binding to a hydrophobic Teflon surface and the ability to self-assemble in the amyloid-like form and to form rodlets were also monitored. DISCUSSIONSC3 is a very stable protein. The CD spectrum of the monomeric form hardly changes in 10 mm DTT or in 5.4 mguanidine (Fig. 2). In addition, heating to 90 °C or changing the pH between 3 and 12 does not change the secondary structure of the protein (data not shown). SC3 only unfolds after reduction of the disulfide bridges with 10 mm DTT in 5.4 m guanidine. This shows that the disulfide bridges are essential for the stability of the monomeric form of SC3.In general the conformation of proteins that adsorb on a hydrophobic solid support varies from a native or nearly native conformation to an extensively unfolded conformation (18Brash J.L. Horbett T.A. Horbett T.A. Brash J.L. Protein at Interfaces II: Fundamentals and Applications. 602. American Chemical Society, Washington, D. C.1995: 1-23Google Scholar, 19Haynes C.A. Norde W. J. Colloid Interface Sci. 1995; 169: 313-328Crossref Scopus (352) Google Scholar). The remarkable refolding of IAA-SC3 on Teflon, with eight additional negative charges, demonstrates the high propensity of the hydrophobin to refold on a hydrophobic surface and is a clear illustration of the specific interaction of hydrophobins with hydrophobic substrates. However, the stability of the IAA-SC3 layer is decreased compared with a layer of assembled native SC3. This instability is not caused by a difference in structure but by the negative charges at pH 7.0, as shown by the recovery of the strong binding when the extraction with SDS was performed at low pH. Furthermore, the IAA-SC3 is still capable of forming rodlets (Fig.7 A). This confirms the observation that intermolecular disulfide bridges are not involved in the self-assembly process.All hydrophobins have the same distribution of cysteine residues, and they probably have the same pattern of disulfide bridges. They are produced as monomeric proteins and can self-assemble into more or less insoluble films after interaction with a hydrophobic-hydrophilic interface. For SC3 it has been shown that this film consists of amyloid fibrils1. For many amyloid proteins the polymerization is initiated by a partially folded state of the protein (20Taubes G. Science. 1996; 271: 1493-1495Crossref PubMed Scopus (131) Google Scholar, 21Kelly J.W. Curr. Opin. Struct. Biol. 1998; 8: 101-106Crossref PubMed Scopus (947) Google Scholar). Therefore it is understandable that opening of the disulfide bridges (which destabilizes the monomeric form) increases the propensity of hydrophobins to form amyloid fibrils.IAM-SC3 in water spontaneously self-assembles in an amyloid-like β-sheet form in the absence of a hydrophobic-hydrophilic interface, but it first goes through a short-lived α-helical state. This corroborates the observation that the α-helical form also in native SC3 is an intermediate in the self-assembly process2. We propose that the disulfide bridges stabilize the monomeric form of hydrophobins and prevent premature self-assembly (before interaction with a hydrophobic-hydrophilic interface), which would result in protein aggregates within the cell or its aqueous environment. The eight conserved cysteine residues in hydrophobins are thus essential for the function of hydrophobins at hydrophobic-hydrophilic interfaces during fungal development. SC3 is a very stable protein. The CD spectrum of the monomeric form hardly changes in 10 mm DTT or in 5.4 mguanidine (Fig. 2). In addition, heating to 90 °C or changing the pH between 3 and 12 does not change the secondary structure of the protein (data not shown). SC3 only unfolds after reduction of the disulfide bridges with 10 mm DTT in 5.4 m guanidine. This shows that the disulfide bridges are essential for the stability of the monomeric form of SC3. In general the conformation of proteins that adsorb on a hydrophobic solid support varies from a native or nearly native conformation to an extensively unfolded conformation (18Brash J.L. Horbett T.A. Horbett T.A. Brash J.L. Protein at Interfaces II: Fundamentals and Applications. 602. American Chemical Society, Washington, D. C.1995: 1-23Google Scholar, 19Haynes C.A. Norde W. J. Colloid Interface Sci. 1995; 169: 313-328Crossref Scopus (352) Google Scholar). The remarkable refolding of IAA-SC3 on Teflon, with eight additional negative charges, demonstrates the high propensity of the hydrophobin to refold on a hydrophobic surface and is a clear illustration of the specific interaction of hydrophobins with hydrophobic substrates. However, the stability of the IAA-SC3 layer is decreased compared with a layer of assembled native SC3. This instability is not caused by a difference in structure but by the negative charges at pH 7.0, as shown by the recovery of the strong binding when the extraction with SDS was performed at low pH. Furthermore, the IAA-SC3 is still capable of forming rodlets (Fig.7 A). This confirms the observation that intermolecular disulfide bridges are not involved in the self-assembly process. All hydrophobins have the same distribution of cysteine residues, and they probably have the same pattern of disulfide bridges. They are produced as monomeric proteins and can self-assemble into more or less insoluble films after interaction with a hydrophobic-hydrophilic interface. For SC3 it has been shown that this film consists of amyloid fibrils1. For many amyloid proteins the polymerization is initiated by a partially folded state of the protein (20Taubes G. Science. 1996; 271: 1493-1495Crossref PubMed Scopus (131) Google Scholar, 21Kelly J.W. Curr. Opin. Struct. Biol. 1998; 8: 101-106Crossref PubMed Scopus (947) Google Scholar). Therefore it is understandable that opening of the disulfide bridges (which destabilizes the monomeric form) increases the propensity of hydrophobins to form amyloid fibrils. IAM-SC3 in water spontaneously self-assembles in an amyloid-like β-sheet form in the absence of a hydrophobic-hydrophilic interface, but it first goes through a short-lived α-helical state. This corroborates the observation that the α-helical form also in native SC3 is an intermediate in the self-assembly process2. We propose that the disulfide bridges stabilize the monomeric form of hydrophobins and prevent premature self-assembly (before interaction with a hydrophobic-hydrophilic interface), which would result in protein aggregates within the cell or its aqueous environment. The eight conserved cysteine residues in hydrophobins are thus essential for the function of hydrophobins at hydrophobic-hydrophilic interfaces during fungal development. We thank Nathalie Sonveaux and Jean-Marie Ruysschaert for assistance in the recording of the attenuated total reflection Fourier transform infrared spectra.