Fractal intermediates in the self-assembly of silicatein filaments

Abstract

Silicateins are proteins with catalytic, structure-directing activity that are responsible for silica biosynthesis in certain sponges; they are the constituents of macroscopic protein filaments that are found occluded within the silica needles made by Tethya aurantia. Self-assembly of the silicatein monomers and oligomers is shown to form fibrous structures by a mechanism that is fundamentally different from any previously described filament-assembly process. This assembly proceeds through the formation of diffusion-limited, fractally patterned aggregates on the path to filament formation. The driving force for this self-assembly is suggested to be entropic, mediated by the interaction of hydrophobic patches on the surfaces of the silicatein subunits that are not found on highly homologous congeners that do not form filaments. Our results are consistent with a model in which silicatein monomers associate into oligomers that are stabilized by intermolecular disulfide bonds. These oligomeric units assemble into a fractal network that subsequently condenses and organizes into a filamentous structure. These results represent a potentially general mechanism for protein fiber self-assembly.

Fig. 1.

Identification of probable sites of subunit–subunit interactions for self-assembly. (a) Hydropathy analysis. Five regions where the hydrophobicity differs between filament forming silicateins (black) and the soluble cathepsins (gray) were identified. The asterisk marks the position at which cathepsin L is glycosylated (51), which lowers the hydrophobicity in that region. Amino acid positions are color-coded as follows: orange, 10–16; blue, 31–42; yellow, 59–61; red, 72–80; pink, 105–123; and green, 151–159. (b) Superimposition of the color-coded regions on a model of silicatein α derived from the crystal structure of cathepsin L shows they are present on the solvent-accessible surface of silicatein.

Fig. 2.

Dissolution of the silicatein filaments. (a) Turbidity measurements taken at 600 nm of filaments before and after addition of agents that disrupt the filament structure. (b) Weight-averaged distribution of light-scattering data with and without DTT, respectively. The data show that one major population exists with an average radius of hydration of 7.0 nm in the absence of DTT and one major population exists with an average radius of hydration of 4.0 nm in the presence of DTT.

Fig. 3.

Transmission electron micrographs of structural intermediates in the self-assembly process at 15 (a), 30 (b), and 60 (c) min. (Scale bars, 100 nm.)

Fig. 4.

Transmission electron micrographs of fractal structures and the calculation of fractal dimension by using the box-counting method. (a and c) The 30-min intermediate. (b and d) The 60-min time point, showing a fractal network flanked by filamentous structures. Fractal dimension was calculated only for the area in the rectangle. (Scale bar, 100 nm.)

Fig. 5.

Salt and pH dependence of aggregation of silicatein oligomers. (a) OD₆₀₀ of oligomers as a function of salt and pH conditions. (b) Scanning electron micrograph of the morphology of the product generated under low-pH, high-salt conditions.

Fig. 6.

Model of fractal assembly. Silicatein monomers associate into oligomers via disulfide bonds. The oligomers form fractal networks by DLA. As soon as the fractal network is formed, the close proximity and reduction in degrees of freedom drives the condensation and organization into a filament.