Molecular Dynamics Study of the Folding of Hydrophobin SC3 at a Hydrophilic/Hydrophobic Interface

Hydrophobins are fungal proteins that self-assemble at hydrophilic/hydrophobic interfaces into amphipathic membranes. These assemblages are extremely stable and posses the remarkable ability to invert the polarity of the surface on which they are adsorbed. Neither the three-dimensional structure of a hydrophobin nor the mechanism by which they function is known. Nevertheless, there are experimental indications that the self-assembled form of the hydrophobins SC3 and EAS at a water/air interface is rich with β-sheet secondary structure. In this paper we report results from molecular dynamics simulations, showing that fully extended SC3 undergoes fast (~100 ns) folding at a water/hexane interface to an elongated planar structure with extensive β-sheet secondary elements. Simulations in each of the bulk solvents result in a mainly unstructured globular protein. The dramatic enhancement in secondary structure, whether kinetic or thermodynamic in origin, highlights the role interfaces between phases with large differences in polarity can have on folding. The partitioning of the residue side-chains to one of the two phases can serve as a strong driving force to initiate secondary structure formation. The interactions of the side-chains with the environment at an interface can also stabilize configurations that otherwise would not occur in a homogenous solution.

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

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.

Assembly of the Fungal SC3 Hydrophobin into Functional Amyloid Fibrils Depends on Its Concentration and Is Promoted by Cell Wall Polysaccharides

Class I hydrophobins function in fungal growth and development by self-assembling at hydrophobic-hydrophilic interfaces into amyloid-like fibrils. SC3 of the mushroom-forming fungus Schizophyllum commune is the best studied class I hydrophobin. This protein spontaneously adopts the amyloid state at the water-air interface. In contrast, SC3 is arrested in an intermediate conformation at the interface between water and a hydrophobic solid such as polytetrafluoroethylene (PTFE; Teflon). This finding prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that SC3 adopts the amyloid state at the water-PTFE interface at high concentration (300 μg ml−1) and prolonged incubation (16 h). Moreover, we show that amyloid formation at both the water-air and water-PTFE interfaces is promoted by the cell wall components schizophyllan (β(1–3),β(1–6)-glucan) and β(1–3)-glucan. Hydrophobin concentration and cell wall polysaccharides thus contribute to the role of SC3 in formation of aerial hyphae and in hyphal attachment

Self-assembly of the hydrophobin SC3 proceeds via two structural intermediates

Hydrophobins self assemble into amphipathic films at hydrophobic–hydrophilic interfaces. These proteins are involved in a broad range of processes in fungal development. We have studied the conformational changes that accompany the self-assembly of the hydrophobin SC3 with polarization-modulation infrared reflection absorption spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism, and related them to changes in morphology as observed by electron microcopy. Three states of SC3 have been spectroscopically identified previously as follows: the monomeric state, the α-helical state that is formed upon binding to a hydrophobic solid, and the β-sheet state, which is formed at the air–water interface. Here, we show that the formation of the β-sheet state of SC3 proceeds via two intermediates. The first intermediate has an infrared spectrum indistinguishable from that of the α-helical state of SC3. The second intermediate is rich in β-sheet structure and has a featureless appearance under the electron microscope. The end state has the same secondary structure, but is characterized by the familiar 10-nm-wide rodlets