Probing Structural Changes during Self-assembly of Surface-Active Hydrophobin Proteins that Form Functional Amyloids in Fungi

doi:10.1016/j.jmb.2018.07.025

Journal of Molecular Biology

Volume 430, Issue 20, 12 October 2018, Pages 3784-3801

https://doi.org/10.1016/j.jmb.2018.07.025 Get rights and content

Highlights

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Class I hydrophobins assemble with conformational change into functional amyloid.
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Heating of hydrophobins can induce the formation of alternative structures.
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Conversion to amyloid involves a shift from right-twisted to more relaxed β-structure.
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Hydrophobin surface activity and plasticity vary with diverse biological functions.

Abstract

Hydrophobins are amphiphilic proteins secreted by filamentous fungi in a soluble form, which can self-assemble at hydrophilic/hydrophobic or water/air interfaces to form amphiphilic layers that have multiple biological roles. We have investigated the conformational changes that occur upon self-assembly of six hydrophobins that form functional amyloid fibrils with a rodlet morphology. These hydrophobins are present in the cell wall of spores from different fungal species. From available structures and NMR chemical shifts, we established the secondary structures of the monomeric forms of these proteins and monitored their conformational changes upon amyloid rodlet formation or thermal transitions using synchrotron radiation circular dichroism and Fourier-transform infrared spectroscopy (FT-IR). Thermal transitions were followed by synchrotron radiation circular dichroism in quartz cells that allowed for microbubbles and hence water/air interfaces to form and showed irreversible conformations that differed from the rodlet state for most of the proteins. In contrast, thermal transitions on hermetic calcium fluoride cells showed reversible conformational changes. Heating hydrophobin solutions with a water/air interface on a silicon crystal surface in FT-IR experiments resulted in a gain in β-sheet content typical of amyloid fibrils for all except one protein. Rodlet formation was further confirmed by electron microscopy. FT-IR spectra of pre-formed hydrophobin rodlet preparations also showed a gain in β-sheet characteristic of the amyloid cross-β structure. Our results indicate that hydrophobins are capable of significant conformational plasticity and the nature of the assemblies formed by these surface-active proteins is highly dependent on the interface at which self-assembly takes place.

Graphical abstract

Section snippets

Introduction and Background

Filamentous fungi produce and secrete small amphipathic proteins, known as hydrophobins, which are important in modulating the interactions between these fungi and their environments [1]. Hydrophobins have the capacity to spontaneously self-assemble at hydrophobic/hydrophilic interfaces (HHIs) to form amphipathic layers. The layers of some hydrophobins, known as class I hydrophobins, consist of robust fibrillar structures known as rodlets, which have an underlying cross-β amyloid structure.

SRCD analyses of hydrophobins in solution reflect the characteristic hydrophobin β-barrel monomer structures, with a significant amount of irregular structure combined with right-hand twisted antiparallel β-sheets

SRCD spectra were collected from the monomeric forms of the six hydrophobins in solution under standard conditions (25 °C, atmospheric pressure) using a quartz glass cell. The data were processed using CDtool [27] and spectral fitting was performed with BeStSel [24], using a linear combination of eight structural components that provided close agreement with experimental data (Fig. 1). The SRCD results are consistent with the shared hydrophobin fold in all hydrophobins, which comprises a small

Conclusions

SRCD was chosen as a method of analysis of hydrophobin structure and conformational plasticity because of the increased informational content arising from the accessible lower wavelengths and the possibility of discriminating between parallel and anti-parallel β-sheets and left-hand twisted, right-hand-twisted and relaxed β-structures. Estimation of the secondary structure content of the monomeric forms of MPG1, DewA, EAS_∆ 15, RodA, RodB and RodC in solution reveals that the hydrophobins from A.

Protein expression and purification

All hydrophobins were produced as fusion proteins with an N-terminal hexahistidine (His₆) tagged ubiquitin (Ub) and a cleavage site between the Ub sequence and the hydrophobin (Hyd) sequence. A similar protocol was used for the six proteins. The H₆-Ub-Hyd proteins were expressed using the Escherichia coli BL21 (DE3) strain. Bacteria were grown at 37 °C, expression was induced with IPTG and cells were harvested by centrifugation after 3 or 4 h of induction. The fusion proteins were (i) extracted

Acknowledgments

This work was supported by the Australian Research Council in the form of Discovery Project Grants to M.S. and A.H.K. (DP120100756 and DP150104227) and by Australian Government funding in the form of Postgraduate Research Awards to V.L. and S.B. The data collection at SOLEIL Synchrotron was funded by proposal 20160949, and we are grateful to Matthieu Réfrégiers for assistance with the application for beam time. We thank and acknowledge the support of the staff, particularly Joonsup Lee, in the

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One type is a hydrophobic protein composed of small rods with the potential to form cross-β structures in the protein fibrils, and the other type is a protein structure without fibrillar structure after self-assembly (Jensen, Andersen, Pedersen, Frisvad, & Søndergaard, 2010). Related scholars also extracted six type I hydrophobin from the spore cell wall of Aspergillus fumigatus, A. nidulans, Magnaporthe oryzae as well as Neurospora crassa, and it was concluded that their conformations were highly plastic and could self-assemble to form rod-shaped protein fibrils, thereby imparting fibrils desirable biological functions that were not provided by the protein monomers (Pham et al., 2018). Compared with natural protein, protein fibrils have higher viscosity and gelling properties, and it is speculated that the increase in viscosity during protein fibrillogenesis may be caused by the formation of tangled dense network structure and the increase in the hydrodynamic diameter of the protein.
In recent years, protein fibrillization has attracted extensive attention on account of endowing food with desirable functional properties and better stability, which makes it a favorable option to be employed in diverse applications. Protein fibrils can be self-assembled under appropriate external conditions and regarded as advanced food materials.
This review elaborates the self-assembly of protein fibrils, the factors affecting protein fibrillogenesis and characterization methods of fibrillation. In particular, the fibrillation conditions of proteins, the techno-functional properties and applications of protein fibrils in the food field are highlighted. Finally, the research trends of protein fibrils have been proposed.
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Hydrophobins are commonly classified as classes I and II depending on their self-assembly form [20–23]. Some of the well-studied hydrophobins include class II hydrophobins HFBI and HFBII from Trichoderma reesei [14,15,24–33], class I hydrophobins EAS from Neurospora crassa [34–36], RodA and DewA from Aspergillus nidulans [37,38], and SC3 from Schyzophyllum commune [39–44]. While class I hydrophobins form amyloid-like rodlet self-assembly structures that are robust [35] and cannot be dissociated by pressure, detergent, or 60 % ethanol [45,46]; class II hydrophobins form regularly packed, ultra-structured monolayers [32] that can easily be dissociated under the same conditions [47].
Hydrophobins are fungal proteins that can mediate water surface tension by forming amphiphilic self-assembly structures in hydrophobic–hydrophilic interfaces. Hydrophobins are known to self-assemble into two forms depending on their class: class I hydrophobins aggregate into a functional amyloid rodlet, while class II hydrophobins aggregate into a regularly patterned monolayer. Owing to its unique properties, hydrophobin has been considered as a biocompatible nanomaterial for various applications and there have been several attempts to engineer hydrophobins to enhance their function. Recently, a chimeric hydrophobin named NChi2 was found to be able to self-assemble into both rodlet and monolayer forms depending on the incubating environment. Although this remarkable feature suggests that NChi2 can function as a versatile bionanomaterial for various applications, only little information about the protein, such as its assembly structure or its characteristics, is provided. To investigate the extraordinary behavior of NChi2, it seems to be a prerequisite to first understand the characteristics of its parent hydrophobins, namely class I EAS and class II NC2. Here, we conducted a preliminary study on predicting the self-assembly structure of class II hydrophobin NC2 and estimating its structural characteristics by employing several computational methods. From the results, we found that NC2 shows stronger surface activity than HFBII, while its assembly structure is weaker than that of HFBII. We hope that this research serves as a foundation to further investigate the structural characteristics of a unique hydrophobin NChi2 in future studies.

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^†: C.L.L.P. and B.R.F. contributed equally to this work.

⁶: Present address: R. Dazzoni, Institute of Chemistry and Biology of Membranes and Nano-Objects, 33607 Pessac, France.

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