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Characterization of a Basidiomycota hydrophobin reveals the structural basis for a high-similarity Class I subdivision

View Article: PubMed Central - PubMed

ABSTRACT

Class I hydrophobins are functional amyloids secreted by fungi. They self-assemble into organized films at interfaces producing structures that include cellular adhesion points and hydrophobic coatings. Here, we present the first structure and solution properties of a unique Class I protein sequence of Basidiomycota origin: the Schizophyllum commune hydrophobin SC16 (hyd1). While the core β-barrel structure and disulphide bridging characteristic of the hydrophobin family are conserved, its surface properties and secondary structure elements are reminiscent of both Class I and II hydrophobins. Sequence analyses of hydrophobins from 215 fungal species suggest this structure is largely applicable to a high-identity Basidiomycota Class I subdivision (IB). To validate this prediction, structural analysis of a comparatively distinct Class IB sequence from a different fungal order, namely the Phanerochaete carnosa PcaHyd1, indicates secondary structure properties similar to that of SC16. Together, these results form an experimental basis for a high-identity Class I subdivision and contribute to our understanding of functional amyloid formation.

No MeSH data available.


SC16 is an active Class I hydrophobin.(A) Linear dependency of enhanced fluorescence of Thioflavin T induced by aeration and protein concentration. Gentle head over tail overnight aeration on a rotary shaker induced enhanced ThT fluorescence. N = 5 for experiment while N = 3 for control (no shaking and no protein). All error bars indicate standard deviation. (B) AFM image of dried down SC16 in water (aerated) on HOPG reveals SC16 assembled into rodlets. Model prepared using Nanoscope software (version 5.12r3) (C) Width, and (D) length distributions indicates SC16 elongates after initial assembly. The average thickness of the layer is 1.99 ± 0.82 nm.
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f1: SC16 is an active Class I hydrophobin.(A) Linear dependency of enhanced fluorescence of Thioflavin T induced by aeration and protein concentration. Gentle head over tail overnight aeration on a rotary shaker induced enhanced ThT fluorescence. N = 5 for experiment while N = 3 for control (no shaking and no protein). All error bars indicate standard deviation. (B) AFM image of dried down SC16 in water (aerated) on HOPG reveals SC16 assembled into rodlets. Model prepared using Nanoscope software (version 5.12r3) (C) Width, and (D) length distributions indicates SC16 elongates after initial assembly. The average thickness of the layer is 1.99 ± 0.82 nm.

Mentions: The solution properties of recombinant SC16 produced in Escherichia coli were consistent with those of other Class I hydrophobins10. Specifically, upon introduction of an air-water interface by gentle head over tail shaking (i.e. aeration) specific binding to the amyloid specific dye ThT was induced as indicated by the linear relationship between enhanced fluorescence of the amyloidogenic dye and protein concentration (Fig. 1A), while the fluorescence emission maxima remained constant at 483 nm regardless of protein concentration (Figure S2). Furthermore, after aeration atomic force microscopy (AFM) revealed the presence of rodlet-shaped assemblies on a dried down droplet of SC16 in water on highly oriented pyrolytic graphite (HOPG) (Fig. 1B). No assemblies were observed by AFM in the absence of aeration prior to drying on HOPG at the deposited concentration tested. The ~ 2 nm height of the rodlet assemblies is similar to that reported for SC3 (2.5–3 nm)24. The distribution of rodlet widths is comparatively narrower than the distribution of lengths and is centred at approximately 5 nm (Fig. 1C,D). The length distribution is centred at approximately 15 nm.


Characterization of a Basidiomycota hydrophobin reveals the structural basis for a high-similarity Class I subdivision
SC16 is an active Class I hydrophobin.(A) Linear dependency of enhanced fluorescence of Thioflavin T induced by aeration and protein concentration. Gentle head over tail overnight aeration on a rotary shaker induced enhanced ThT fluorescence. N = 5 for experiment while N = 3 for control (no shaking and no protein). All error bars indicate standard deviation. (B) AFM image of dried down SC16 in water (aerated) on HOPG reveals SC16 assembled into rodlets. Model prepared using Nanoscope software (version 5.12r3) (C) Width, and (D) length distributions indicates SC16 elongates after initial assembly. The average thickness of the layer is 1.99 ± 0.82 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385502&req=5

f1: SC16 is an active Class I hydrophobin.(A) Linear dependency of enhanced fluorescence of Thioflavin T induced by aeration and protein concentration. Gentle head over tail overnight aeration on a rotary shaker induced enhanced ThT fluorescence. N = 5 for experiment while N = 3 for control (no shaking and no protein). All error bars indicate standard deviation. (B) AFM image of dried down SC16 in water (aerated) on HOPG reveals SC16 assembled into rodlets. Model prepared using Nanoscope software (version 5.12r3) (C) Width, and (D) length distributions indicates SC16 elongates after initial assembly. The average thickness of the layer is 1.99 ± 0.82 nm.
Mentions: The solution properties of recombinant SC16 produced in Escherichia coli were consistent with those of other Class I hydrophobins10. Specifically, upon introduction of an air-water interface by gentle head over tail shaking (i.e. aeration) specific binding to the amyloid specific dye ThT was induced as indicated by the linear relationship between enhanced fluorescence of the amyloidogenic dye and protein concentration (Fig. 1A), while the fluorescence emission maxima remained constant at 483 nm regardless of protein concentration (Figure S2). Furthermore, after aeration atomic force microscopy (AFM) revealed the presence of rodlet-shaped assemblies on a dried down droplet of SC16 in water on highly oriented pyrolytic graphite (HOPG) (Fig. 1B). No assemblies were observed by AFM in the absence of aeration prior to drying on HOPG at the deposited concentration tested. The ~ 2 nm height of the rodlet assemblies is similar to that reported for SC3 (2.5–3 nm)24. The distribution of rodlet widths is comparatively narrower than the distribution of lengths and is centred at approximately 5 nm (Fig. 1C,D). The length distribution is centred at approximately 15 nm.

View Article: PubMed Central - PubMed

ABSTRACT

Class I hydrophobins are functional amyloids secreted by fungi. They self-assemble into organized films at interfaces producing structures that include cellular adhesion points and hydrophobic coatings. Here, we present the first structure and solution properties of a unique Class I protein sequence of Basidiomycota origin: the Schizophyllum commune hydrophobin SC16 (hyd1). While the core β-barrel structure and disulphide bridging characteristic of the hydrophobin family are conserved, its surface properties and secondary structure elements are reminiscent of both Class I and II hydrophobins. Sequence analyses of hydrophobins from 215 fungal species suggest this structure is largely applicable to a high-identity Basidiomycota Class I subdivision (IB). To validate this prediction, structural analysis of a comparatively distinct Class IB sequence from a different fungal order, namely the Phanerochaete carnosa PcaHyd1, indicates secondary structure properties similar to that of SC16. Together, these results form an experimental basis for a high-identity Class I subdivision and contribute to our understanding of functional amyloid formation.

No MeSH data available.