A Structural and Functional Role for Disulfide Bonds in a Class II

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A structural and functional role for disulfide bonds in a class II hydrophobin Nathanael D Sallada, Kerri J Dunn, and Bryan W Berger Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01166 • Publication Date (Web): 26 Dec 2017 Downloaded from http://pubs.acs.org on December 27, 2017

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A structural and functional role for disulfide bonds in a class II hydrophobin Nathanael D. Sallada§, Kerri J. Dunn†, Bryan W. Berger*†§ †Department of Chemical and Biomolecular Engineering, Lehigh University, 111 Research Drive, Bethlehem, Pennsylvania 18015, United States §Department of Bioengineering, Lehigh University, 111 Research Drive, Bethlehem, Pennsylvania 18015, United States *Phone: (434) 924-2016, E-mail: [email protected]

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Abstract

Hydrophobins are multifunctional, highly surface-active proteins produced in filamentous fungi and are identifiable by 8 conserved cysteine residues, which form 4 disulfide bridges. These proteins can be subdivided into two classes based on their hydropathy profiles, solubility, and structures formed upon interfacial assembly. Here, we probe the structural and functional roles of disulfide bonds for a class II hydrophobin in different interfacial contexts by reducing its disulfides with 1,4-dithiothreitol and blocking the free thiols with iodoacetamide, then examining protein secondary structure, emulsification capability, hydrophobic surface wetting, and solution self-assembly. Changes in circular dichroism spectra upon reduction and blocking of disulfides are consistent with an increase in random coil secondary structure. Emulsification of octane in water using reduced and unreduced forms of class II hydrophobin showed a substantial loss of emulsification ability without disulfides and stable emulsion formation for hydrophobin with disulfides. Additionally, water contact angle measurements done on polytetrafluoroethylene treated with solutions of reduced and unreduced hydrophobin showed efficient wetting of the hydrophobic surface for unreduced samples only. Lastly, Förster Resonance Energy Transfer (FRET) was used to assess the role of disulfides on self-assembly in solution, and near complete loss of FRET signal is consistent with a model in which solution self-assembly does not occur after reduction and blocking of the disulfides. From this we conclude that, in contrast to class I hydrophobins, the disulfides of this class II hydrophobin are required for protein structural stability, surface activity at both liquid-liquid and solid-liquid interfaces, and solution selfassembly.

Keywords: hydrophobin, biosurfactant, self-assembly, disulfide


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Introduction Hydrophobins are small, highly surface active proteins secreted by filamentous fungi1. Hydrophobins are identifiable by a characteristic pattern of 8 cysteine residues, which form 4 intermolecular disulfide bonds1. These proteins play various roles in fungal development and morphogenesis, specifically in processes that occur at hydrophobic-hydrophilic interfaces such as in coating of aerial structures and spores, lowering water surface tension, and aiding in attachment to surfaces2,3. Their surface active properties, resistance to degradation, and immunological inertness have made them attractive candidates for many applications such as protein purification4, increasing implant biocompatibility, increasing water solubility of insoluble drugs5,6, and foam stabilizers for food products7.

Based on their hydropathy plots, their

solubility, and the structures they form at interfaces, two classes of hydrophobins have been identified (class I and class II) 2,3. Class I hydrophobins self assemble at hydrophobic-hydrophilic interfaces into amphipathic membranes composed of amyloid-like rodlets which are highly stable and only dissolvable in harsh acids, while class II hydrophobins form monolayers which are easily dissolved with organic solvents and detergents1,6,8,9. The crystal structures of Trichoderma reesei class II hydrophobins HFBI and HFBII have been solved, each revealing the presence of a large, flat hydrophobic patch on an otherwise hydrophilic surface10,11. These structures indicate the nature of the hydrophobins’ amphiphilicity and inform how these hydrophobins may function. In the case of the class II hydrophobins HFBI and HFBII, solution multimerization is believed to be mainly driven by the hydrophobic effect12, whereby self assembly in solution occurs in a concentration dependent manner in order to sequester their hydrophobic patches to enhance their solubility, but additional work suggests there are specific molecular interactions between 3 ACS Paragon Plus Environment

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different hydrophobins in solution that stabilize multimers, as a study of dynamics in class II hydrophobin multimer exchange for mixtures of HFBI and HFBII resulted in lowering of activation energy and half-life of exchange compared to HFBII alone, but addition of other surface active proteins had no such effect13. Furthermore, in studies considering the preference of HFBI to multimerize in solution versus adsorb at an air-water interface, an engineered HFBI variant with a higher propensity for solution multimer formation did not noticeably alter its ability to assemble at an interface14. Similar to the case of solution multimerization, this and other studies suggest specific protein-protein interactions within class II hydrophobins are important for surface assembly into monolayers, as surface assembled HFBI and HFBII exhibited different arrangements of molecules in the unit cell when studied by grazing incidence X-ray diffraction in situ, and mutations to specific, charged residues on the HFBI surface affected the viscoelastic properties of formed films rather than film assembly15,16. Thus, specific residues and interfaces within class II hydrophobins are important for both assembly in solution and at an interface, with specific molecular determinants of both remaining an active area of research. Given the conservation of 8 cysteine residues in virtually all hydrophobins (class I and II), these residues have naturally prompted research into their structural and functional roles in both multimer and interfacial assembly. de Vocht et al. concluded after reducing and chemically blocking the disulfide bonds of class I hydrophobin SC3 from Schizophyllum commune that these disulfide bridges are not required for self assembly, but function to keep monomers soluble until they can adsorb to an interface and assemble into rodlet ultrastructures, thus keeping the proteins in an active state by preventing self-assembly in solution17. In line with the findings of de Vocht et al., Kershaw et al. found that removal of disulfide bonds from the hydrophobin MpgI, a class I


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Biochemistry

hydrophobin18, did not affect its ability to self-assemble in the fungal cell, but hindered its extracellular secretion and localization in the cell wall19. Interestingly in contrast to these findings, Linder et al. noted a complete loss of functionality in the class II hydrophobin HFBI after disulfide reduction in the context of an aqueous two phase system (ATPS) separation using nonionic surfactants, in which the unreduced form could be effectively separated into the surfactant phase20. However, it is unclear whether this finding is robust in relation to class II hydrophobin functionality in different interfacial contexts, given that Linder et al. also noted the class II hydrophobin HFBI does not partition into other hydrophobic solvents such as isobutanol, and so more complex structural features outside of purely hydrophobic affinity must be involved in the surfactant interaction20. Due to the multifunctionality of hydrophobins in nature, interfacial context is an important parameter to consider when trying to understand hydrophobin structurefunction relationships, as they are implicated in solid surface attachment, lowering water tension at air-water interfaces, and in producing protective layers on fungal cell walls2,3, which are all different interfaces and therefore present different driving forces. Moreover, how the loss of functionality after reduction for class II hydrophobins relates directly to class II hydrophobin structural stability remains to be rigorously elucidated, since in the case of class I hydrophobin SC3 there is a disconnect between protein structural stability governed by disulfide bonds and amphiphilic functionality17. Given that the two hydrophobin classes have markedly different surface properties, very low sequence similarity outside of the 8 conserved cysteine residues, and are not necessarily found in the same fungal phyla21, it is therefore of interest to determine what role disulfides play in class II hydrophobin structure, function and assembly in different interfacial contexts that are pertinent to possible applications of these biosurfactants, namely at liquid-liquid and solid-liquid


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interfaces. This is important in order to understand the disulfide bond dependence of class II structure-function relationships, whether these relationships are robust or dependent on different interfacial contexts, and how disulfide dependent structure-function relationships in class II compare to those of class I hydrophobins. In this study, we examine the roles of disulfide bonds of a recombinantly produced class II hydrophobin in structural stability, interfacial assembly at liquid and solid interfaces, and solution self-assembly in order to delineate fundamental structure-function relationship differences between class I and class II hydrophobins as they relate to structure and function in different interfacial contexts. Experimental Section Gene Synthesis and Cloning. Unless otherwise stated, standard molecular biology procedures were used for DNA preparation and subcloning. The pPICZα A expression vector (Invitrogen) was digested with NotI and XhoI in Buffer 3.1 (New England Biolabs (NEB)) for 4 h at 37oC. Digested plasmid was purified by gel extraction using GenElute Gel Extraction Kit (Sigma). The hydrophobin HFBI primary sequence from T. reesei (Uniprot P52754) was used as the basis for a recombinant N-terminal hexahistidine (His6)-hemaglutinnin (HA) tagged hydrophobin gene. The hydrophobin gene was synthesized by assembly-PCR using oligonucleotides fragments corresponding to the full-length gene (Integrated DNA Technologies) and introduced a Phe-Trp mutation (corresponding to Phe13 in the native mature sequence of HFBI) to determine protein concentration through measurement of intrinsic Trp absorbance at 280 nm. Final PCR on this product added a C-terminal stop codon and NotI cleavage site, and also added an N-terminal XhoI cleavage site and rebuilt the Kex2 recognition sequence to the arginine codon at nucleotides 1193-1195 to allow for flush insertion of the gene N-terminal amino acid downstream of the EAEA repeats of the backbone. This PCR product was ethanol


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precipitated, reconstituted in nuclease free water (NEB), and doubly digested with XhoI/NotI and purified by gel extraction. The digested vector was added to gene insert in a 1:5 mass ratio and ligated overnight in a 16oC water bath using T4 DNA Ligase (NEB). Ligation was desalted on 0.022 µm nitrocellulose membrane (Millipore) for 1 h before being transformed into E. coli strain DH5α by electroporation. Plasmid was purified from Zeocin resistant colonies using Qiagen Miniprep kit, and the sequence was confirmed by Genscript using 5AOXI standard primer. Yeast Transformation. The pPICZα A vector alone or vector containing the hydrophobin gene was cut using SacI restriction enzyme in buffer 1.1 (NEB). Complete digestion was confirmed by DNA gel electrophoresis and the digested DNA was ethanol precipitated and reconstituted in nuclease free water (NEB) at a concentration of 500 ng/µL. Electrocompetent Pichia pastoris strain GS115 yeast cells were prepared using the protocol described by Wu and Letchworth22 with the following alterations. Yeast was grown in 2 mL YPD (1% yeast extract, 2% tryptone, and 1% dextrose) from a glycerol stock at 28.0oC overnight, and 100 µL of this seed culture was used to inoculate 100 mL of fresh YPD in a 1 L baffled flask. Culture was allowed to grow at 28.0oC at 250 rpm until OD600 reached between 12. The cell pellet was collected at 2,500 g and resuspended in LiAc-DTT buffer (100 mM lithium acetate (LiAc), 10 mM 1,4-dithiothreitol (DTT), 0.6 M sorbitol, 10 mM Tris pH 7.5) and allowed to incubate at room temp for 30 min. Cells were centrifuged at 2,500 g and pellet was washed twice with the same volume of cold 1 M sorbitol and resuspended in cold 1 M sorbitol at a concentration of 1010 cells/mL. 5 µg of linearized plasmid was mixed with 100 µL of prepared GS115 cells in a 2 mm electroporation cuvette. After 5 minute incubation on ice, a 1.5 kV pulse was applied to the cells using a BioRad GenePulser Xcell electroporator and 1 mL of ice cold 1


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M sorbitol was immediately added to the cell mixture. After a 1 h recovery at 30oC without shaking, cells were plated onto YPDS plates of varying Zeocin concentration (1% yeast extract, 2% peptone, 2% dextrose, 1 M sorbitol, 2% agar, 100-1000 µg/ml Zeocin) according to the protocol set forth by Invitrogen. Plates were incubated at 30oC for 3-7 days, until colonies appeared. High-Throughput Selection of High Expression Clones. Colonies from each Zeocin-YPDS plate were selected and inoculated into 400 µL of MGY medium (1% yeast extract, 2% peptone, 1.34% YNB, 4 × 10-5% biotin, 1% glycerol) in a 2 mL deep 96-well PlateOne plate with square wells (USA Scientific), and the top sealed with a sterile AirPore sheet (Qiagen). Several colonies from the empty-vector transformation were also grown as negative expression controls. Plates were grown overnight at 28oC at 400 rpm or until cultures reached saturation. 20 µL of saturated culture was used to inoculate 400 µL fresh MGY in another 96 well plate, and was allowed to grow to saturation as before to normalize cell density between colonies. Plates were spun at 4000 rpm to pellet the cells, and the supernatants carefully removed. To induce protein expression, 400 µL of MMY media (1% yeast extract, 2% peptone, 1.34% YNB, 4 × 10-5% biotin, 1% methanol) was added to each well and the plates were grown overnight as before. Plates were centrifuged to pellet cells, and 3 µL of supernatant was applied to nitrocellulose membrane (Thermo). The membrane was incubated in block solution (5% fat-free dry milk in Tris Buffered Saline + 0.1% Tween 20 (TBS-T)) for 1 h at room temperature, and incubated overnight at 4oC with HRP-conjugated rabbit anti-HA tag antibody (Abcam) diluted 1:1000 in block solution. The membrane was washed 5 times for 5 minutes each in TBS-T, and incubated with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) for 5 minutes at room temperature,


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Biochemistry

and then imaged using a BioRad ChemiDoc MP Imaging System. The clone giving the highest intensity spot was selected for protein production. Expression and Purification. The highest expressing yeast clone was streaked onto a YPD plate, and a single colony was inoculated into 5 mL of fresh MGY in a 50 mL conical tube with the lid taped on to allow for gas exchange. The culture was grown at 28oC @ 250 rpm overnight. 1 mL of this seed culture was used to inoculate 250 mL of fresh MGY, which was grown at 28oC @ 250 rpm until the OD600 reached between 2-6. The cells were pelleted at 2,500 g and the pellets resuspended in 250 ml MMY to an OD600 of 1.0. The cultures were grown as before and 100% methanol was added daily to a final concentration of 1% for 3 days. A colony from the empty-vector transformation was also grown as a negative expression control. After 3 days of induction, the culture was pelleted and the supernatant collected. 100% ethanol was added to the foam adherent to sides of the culture flasks in order to collapse and collect the hydrophobin-containing foam. The foam fraction was centrifuged at 12,000 rpm and filtered with a 0.22 µm filter to remove cells and insoluble material, and then added back to the supernatant. Chelating Sepharose Fast Flow resin (GE) was charged with Ni2+ as per the manufacturer’s instructions, and the culture supernatant was applied to the column at a rate of 2 ml/min using a Biologic LP system (BioRad). The column was washed with 5 column volumes of 20 mM TrisHCl pH 8, followed by 2 column volumes each of 10 mM imidazole, 50 mM imidazole, 150 mM imidazole, and 500 mM imidazole buffers containing 20 mM Tris-HCl pH 8. Fractions were run on 16% Tris-tricine SDS-PAGE under reducing conditions23, and either transferred to nitrocellulose membrane using standard protocols for Western Blot analysis with HRPconjugated rabbit anti-HA tag antibody (Abcam), or directly stained using Coomassie Blue stain.


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Purified hydrophobin fractions were pooled, dialyzed exhaustively against milliQ water and lyophilized or stored as aqueous suspensions for further use. Reduction and Blocking of Cysteine Thiols. 15 ml of 50 µg/ml hydrophobin in 75 mM TrisHCl pH 8 was treated with 20 mM iodoacetamide (IAM)/+2 mM dithiothreitol (DTT), 20 mM IAM/-2 mM DTT only, or no treatment control as follows. 30 µL of 1 M DTT in milliQ water was added to the +DTT sample, and 30 µL of milliQ water was added to the other two conditions. All tubes were incubated at 37oC for 20 minutes, and then 1.5 mL of freshly prepared 1 M proteomic grade IAM stock solution (Alpha Aesar) in 75 mM Tris-Cl pH 8 was added to +DTT and –DTT samples, and milliQ water was added to the no treatment sample. After 15 min incubation at room temperature, the samples were dialyzed against milliQ water at 4oC. Sample molecular weights were analyzed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectroscopy using 10 mg/mL sinapinic acid in 50% acetonitrile + 0.1% TFA as matrix and a Microflex MALDI-TOF mass spectrometer (Bruker). Circular Dichroism (CD) Measurements. Lyophilized IAM/-DTT, IAM/+DTT, and untreated hydrophobin samples were diluted to 10 µM in 10 mM phosphate buffer pH 7. CD was measured in a 0.1 cm quartz cuvette using a Jasco J-815 CD from 240-190 nm at 10 nm/min scan speed, 4 s integration time, and 1 nm bandwidth at 25oC. Reported spectra are averages of 5 scans. Emulsion Production and Dynamic Light Scattering (DLS). Lyophilized IAM/+DTT and IAM/-DTT treated hydrophobin samples were reconstituted in autoclaved milliQ water to a concentration of 10 µM. 50 µL of Oil Red O saturated octane was added to 500 µL of hydrophobin solution in a 2 mL Eppendorf microfuge tube and sonicated on ice with a Misonix Sonicator 3000 tip sonicator on continuous mode, power setting 4.5 for 10 minutes. Oil Red O is a highly lipophilic dye that does not localize to the aqueous phase24. The emulsions were then


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Biochemistry

centrifuged at 3000 rpm for 30 seconds to induce phase separation, and the bottom phase was carefully collected with a pipette and moved to a 2 mL glass vial for stability observation. 50 µL of the IAM/-DTT stabilized emulsion was added to 500 µL of 0.22 µm filtered milliQ water in a DLS cuvette, and DLS autocorrelation function was recorded using an ALV/LSE5004 Light Scattering Electronics and Multiple Tau Digital Correlator (ALV, Germany) at a 90o collection angle at 22oC. The droplet hydrodynamic radius distribution was calculated using an intensity weighted regularized fit in the ALV-7004 Correlator Software version 3.0 (ALV, Germany). Water Contact Angle (WCA). Thoroughly cleaned polytetrafluoroethylene sheets (VWR) were incubated overnight at room temperature in either 10 µM IAM/+DTT or IAM/-DTT treated hydrophobin. The excess protein solution was removed with a pipette and the sheets washed 3 times for 5 minutes each in milliQ water, and then allowed to air dry. 5 µL drops of milliQ water were dispensed on each sheet. The contact angles of the droplets were measured from droplet profile images in ImageJ using the Contact Angle plugin available from NIH25. Förster Resonance Energy Transfer (FRET). Water-soluble sulfo-cyanine 5 (Cy5) and sulfocyanine 3 (Cy3) NHS esters were purchased from Lumiprobe. Lyophilized IAM/-DTT and IAM/+DTT hydrophobin samples were reconstituted to 200 µg/ml in milliQ water, and spun at 12000 rpm for 5 minutes to pellet insoluble material. Supernatant concentration was checked using a NanoDrop 2000 Spectrophotometer (Thermo) and ProtParam calculated extinction coefficients26 at 280 nm of 11960 M -1 cm-1 for IAM/-DTT, and 11460 M -1 cm-1 for IAM/+DTT. 500 µL of each protein solution was adjusted to 0.1 M sodium bicarbonate using a 1 M stock to increase the pH to between 8.3 and 8.5, and then treated at 4oC overnight in the dark with either Cy3 or Cy5 according to the manufacturer’s instructions for mono-labelling. After incubation,


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unincorporated dye was removed by dialysis against milliQ water at 4oC. Degree of labeling was calculated from the ratio of concentration of dye to concentration of protein in the sample. Extinction coefficients used for Cy3 and Cy5 dye concentration determination were respectively 150,000 M -1 cm-1 (549 nm) and 250,000 M -1 cm-1 (647 nm). For FRET experiments, total protein concentration for each sample was 4.5 µM in 10 mM phosphate buffer at pH 7. In the donor-acceptor samples, a 1:1 ratio of Cy3:Cy5 labeled protein was used, and in donor or acceptor only solutions, 2.25 µM of the same unlabeled protein was included to keep the protein concentration constant between samples. Samples were excited at 516 nm (donor excitation), and donor and acceptor emission spectra were measured from 525 nm to 700 nm using a steady-state PTI QuantaMaster 400 fluorescence spectrophotometer (Horiba Scientific). Results and Discussion Methanol induced expression of control (empty-vector transformant) and high hydrophobin expression yeast strains resulted in marked differences in the stability of the foam generated during shaking. Specifically, control strain culture (Figure 1b) produced unstable foam that broke down significantly after 10 minutes of standing at room temperature. In contrast, the high hydrophobin expression strain produced dense foam with substantially greater stability (Figure 1c). Specifically, the hydrophobin-containing foam did not noticeably change after 10 minutes of standing, and remained stable after letting the flask stand for extended times greater than 24 h at room temperature. Therefore, the overproduction and stability of stable foam during cultivation observed (Figures 1b and 1c) indicates proper functionality of the engineered, recombinant hydrophobin, and is consistent with previous work in which overproduction of active hydrophobin HFBI yields stable foams which did not dissipate for three weeks at room

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temperature27,28. Tricine-SDS-PAGE analysis of the high hydrophobin expression cell-free culture supernatant and culture foam (Figure 1d) revealed the presence of a major product at approximately 10 kDa in the foam sample, which is consistent with the expected MW of 9.49 kDa, as well as in the 50 mM and 150 mM imidazole chromatography elutions from affinity purification. Western blot analysis on these same samples using HRP-conjugated rabbit anti-HA tag antibody (Figure 1e) confirm the observed product in the foam and eluate fractions contain the HA tag epitope and therefore are specific to the recombinant hydrophobin fusion protein. Thus, expression and proper secretion of this heterologously produced hydrophobin fusion protein was successful, and addition of N-terminal His6 and HA tags did not affect its functionality as a surface-active protein.


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Figure 1. (a) Primary amino acid sequence of the recombinant class II hydrophobin with His6 and HA tags labeled and underlined, and conservative F-W substitution underlined (b) pPICZ alpha empty vector transformed GS115 expression control after 10 minutes without shaking, in which no significant foam formation occurs (c) High hydrophobin expressing strain of GS115 after 10 minutes without shaking, in which significant foam formation occurs, indicative of hydrophobin overexpression and secretion (d) Coomassie blue stained 16% tricine-SDS-PAGE, 1. molecular weight markers, 2. hydrophobin culture supernatant, 3. hydrophobin culture foam, 4. 50 mM imidazole elution, 5. 150 mM imidazole elution. The expected molecular weight for the recombinant hydrophobin product is 9.49 kDa, which is consistent with the observed major


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product near 10 kDa. (e) anti-HA tag Western Blot of SDS-PAGE samples in (d) confirm the observed product band is specific to the recombinant hydrophobin.

MALDI-TOF analysis of untreated, IAM/-DTT treated, and IAM/+DTT treated hydrophobin (Table 1) reveal that the hydrophobin fusion protein was correctly processed and secreted at the correct size (plus one sodium ion) without glycosylation, and that treatment of hydrophobin with IAM alone did not cause significant change in the protein molecular weight compared to the untreated sample. However, treatment of hydrophobin with IAM/+DTT caused a shift of 464 Da, corresponding to the addition of 8 acetamide groups. Since IAM will react with free thiols29, formation of acetamide-thiol adducts only in the presence of DTT implies that the 8 cysteine residues are otherwise oxidized as disulfide bonds, therefore confirming both the proper formation of the hydrophobin disulfides in the heterologous system, and complete reduction and blocking of these disulfides in the IAM/+DTT sample. Table 1. Molecular weights of hydrophobin samples determined by MALDI-TOF Sample

Molecular Weight (Da)

Untreated Hydrophobin

9511.33

IAM/-DTT Hydrophobin

9513.16

IAM/+DTT Hydrophobin

9975.73

Circular dichroism spectra of untreated, IAM/-DTT, and IAM/+DTT hydrophobin show a clear change in the secondary structure, with loss of signal intensity at 210 nm and a shift in the spectral minimum to lower wavelengths observed for IAM/+DTT hydrophobin only, consistent with an increase in random coil, while insignificant differences are observed between untreated


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and IAM/-DTT hydrophobin samples (Figure 2). These data suggest that the disulfides are key structural features of this hydrophobin and their reduction results in a loss of hydrophobin secondary structure. de Vocht et al. reported a similar loss of secondary structure for the class I Schizophyllum commune hydrophobin SC3 that had been treated with IAM/+DTT, but interestingly the IAMSC3 gained a defined, beta-sheet secondary structure after heating from 4oC to 25oC, and then proceeded to precipitate from solution17. In this study, no such changes were observed in the case of IAM/+DTT treated class II hydrophobin. While in previous studies the class I hydrophobin SC3 refolds into its native structure even after reduction and chemical blockage of its cysteines, in the current study recombinant class II hydrophobin requires intact disulfide bonds for structural stability and does not recover structure after they have been reduced and chemically blocked. Therefore, this study provides direct evidence that despite the sequence conservation of 8 cysteines across class I and class II hydrophobins, their functional consequences in terms of folding and assembly are distinct.


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Figure 2. Circular dichroism spectra of untreated hydrophobin (___), IAM/-DTT hydrophobin (— —), and IAM/+DTT hydrophobin (----). All samples are 10 µM in 10 mM phosphate buffer pH 7.


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Figure 3. Hydrophobin stabilized Oil-Red-O stained octane emulsions. (a) 1. time, t = 0 days, 10 µM IAM/+DTT hydrophobin bottom phase, 2. t = 0 days, 10 µM IAM/-DTT hydrophobin bottom phase. (b) IAM/-DTT hydrophobin bottom phase at t = 7 days. (c) dynamic light scattering hydrodynamic radius intensity weighted distribution of IAM/-DTT hydrophobin emulsion at t = 0 days (---) and at t = 7 days (__).

Oil red O stained-octane emulsions prepared with IAM/-DTT or IAM/+DTT hydrophobin had substantial differences in their bulk solution phase behavior and emulsion stability. Notably, IAM/+DTT was unable to emulsify octane in water to any significant degree, resulting in a clear bottom phase (Figure 3a, 1.). In contrast, IAM/-DTT hydrophobin, with intact disulfide bonds, formed a red bottom phase indicating dispersed octane droplets (Figure 3a, 2.). After 7 days standing at room temperature, there was no obvious phase separation (Figure 3b). Dynamic light scattering of the IAM/-DTT hydrophobin-octane emulsion initially (at 0 days) and at 7 days (Figure 3c) measured a mean hydrodynamic radius of 96.43 nm with a polydispersity index (PD.I.) = 0.105 and hydrodynamic radius of 92.77 nm with a PD.I. = 0.126, respectively. These data are consistent with observation of the bulk solution, in which the IAM/-DTT hydrophobinoctane emulsion is stable for at least one week at room temperature, since the mean droplet size did not change substantially and no visible phase separation occurred. Previous characterization of the class II hydrophobin HFBI indicated stable oil-in-water emulsion formation for at least 3 days using food grade soy oil in a distilled water-hydrophobin suspension7. Here, we show the emulsification properties of HFBI depends on the proper formation of disulfide bonds, as reduction of these bonds results in complete loss in emulsification ability. Thus, as with the observed loss of secondary structure (Figure 2), disulfide bond formation in this recombinant


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class II hydrophobin fusion protein is critical for emulsification and assembly at a hydrophobic oil-water interface. This is consistent with the findings of Linder et al. in the ATPS context, where reduced HFBI completely lost affinity for the nonionic surfactant phase20, but a major difference is that the hydrophobin in the context of emulsions is stabilizing an oil-water interface rather than partitioning into structured micelles as in the ATPS system20, and so the disulfide dependent functionality of this class II hydrophobin in the context of liquid-liquid interface stabilization is robust with regards to disulfide dependent interactions with nonionic surfactants. Water contact angle measurement on PTFE sheets incubated with either IAM/-DTT or IAM/+DTT revealed differences in wettability at a hydrophobic solid interface (Figure 4). Specifically, IAM/-DTT hydrophobin treated PTFE exhibited much higher wettability (WCA = 64.8o ± 1.3o) than the PTFE no protein control (WCA = 116.4o ± 2.0o). IAM/+DTT hydrophobin incubation resulted in an insignificant change in wettability (WCA = 117.6o ± 1.6o) compared to the PTFE no protein control. A previous study using hydrophobin HFBI measured water contact angle values of 59o ± 13o on Teflon30, which is consistent with that of IAM/-DTT treated HFBI in this study and further confirmation that overexpressed HFBI exists in a functionally active state (Figure 1). Furthermore, removal of disulfide bonds prevents wetting of the hydrophobic PTFE interface, as evidenced by the significantly lower contact angle measured after treatment with IAM/+DTT. Collectively, these results indicate hydrophobin HFBI forms an amphipathic film at the hydrophobic PTFE-water interface, and the wettability of hydrophobic PTFE by HFBI is dependent upon the presence of its disulfide bonds. Again, this is consistent with the findings of Linder et al. 20 and our previous emulsification results, which all show that the disulfide bonds of this class II hydrophobin are critical for interfacial self-assembly in liquid-liquid and liquidsolid contexts. Thus, in contrast to class I hydrophobins, class II hydophobin structure and


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function are dependent on the conserved disulfide bonds and environmental context, i.e. liquidliquid or liquid-solid interfaces, have no effect on the hydrophobin’s functionality as an amphiphile when disulfides have been reduced.

Figure 4. Water contact angle measurements on polytetrafluoroethylene treated with 10 µM IAM/-DTT hydrophobin, 10 µM IAM/+DTT hydrophobin, or no protein control. n = 4 for all samples.


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Figure 5. Förster Resonance Energy Transfer (FRET), (a) 2.25 µM Cy3-IAM/-DTT hydrophobin + 2.25 µM unlabeled IAM/-DTT hydrophobin (----), 2.25 µM Cy3-IAM/-DTT + 2.25 µM Cy5-IAM/-DTT (___), 2.25 µM Cy5-IAM/-DTT + 2.25 µM unlabeled IAM/-DTT hydrophobin (…..). (b) 2.25 µM Cy3-IAM/+DTT hydrophobin + 2.25 µM unlabeled IAM/+DTT hydrophobin (----), 2.25 µM Cy3-IAM/+DTT + 2.25 µM Cy5-IAM/+DTT (___), 2.25 µM Cy5IAM/+DTT + 2.25 µM unlabeled IAM/+DTT hydrophobin (…..). Excitation wavelength was 516 nm for all samples. Association of fluorescently labeled IAM/+DTT or IAM/-DTT hydrophobin monomers was studied by FRET in order to gain insight into the changes in tertiary structure and assembly that occur dependent upon disulfide bond formation. FRET depends strongly on the distance between the donor and acceptor fluorophores31, and thus allows for observation of multimerization between differently labeled monomers by observing changes in donor and acceptor emission


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spectra when the donor is excited. FRET-based methods have been used previously to probe class II hydrophobin dynamics and multimerization in solution13,32. Figure 5a shows emission spectra of Cy3-(IAM/-DTT) hydrophobin, Cy5-(IAM/-DTT) hydrophobin, and an equimolar mixture of the two. There is a decrease in the Cy3 emission at 549 nm and increase in Cy5 emission at 675 nm when both fluorophore labeled proteins are present in solution. Figure 5b shows similar spectra for labeled IAM/+DTT hydrophobin, but in this case mixing the separately labeled proteins does not affect the emission spectra of either the donor or the acceptor fluorophores, implying FRET no longer occurs when disulfide bonds are absent. The loss of FRET signal that occurs when disulfides are reduced and blocked is consistent with disulfide bonds being essential to HFBI multimerization in solution. This observation is in direct contrast with what is known regarding the role of disulfides in class I hydrophobin multimerization, where class I hydrophobins do not lose their ability to self-assemble in solution when disulfides are either reduced and chemically blocked, as was the case for hydrophobin SC317, or completely eliminated by site directed mutagenesis, as was the case for hydrophobin Mpg119. Moreover, as specific protein-protein interactions outside of the simple hydrophobic effect have been implicated in class II hydrophobin multimerization13, these data indicate that those sites must also be stabilized by the disulfide bonds since no observable self-interaction occurs upon disulfide reduction. The differences in roles of disulfide bonds between classes could be attributed to other structural differences between the classes, which may have a compensatory effect on stability and functionality for class I, but not for class II. Notably, the loop between the Cys3 and Cys4 positions comprises part of the hydrophobic patch and is much longer and with less conservation in loop length for class I than for class II1. This loop has been implicated in the formation of


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rodlets for the class I hydrophobin HGFI, where substitution of the loop with that of the class II hydrophobin HFBI resulted in loss of rodlet formation upon assembly at the air-water interface33. Moreover, the increased length of this loop region in class I hydrophobins compared to class II hydrophobins allows for large conformational changes to occur upon assembly that are not seen for class II hydrophobins1. This structural freedom in class I hydrophobins could suggest one possible cause for differences in disulfide bond roles between the classes, as this domain could function synergistically with the disulfide bonds in class I hydrophobins to maintain structure and function, while this would not happen in class II structures due to the short loop length. Additionally, it is interesting to note from an evolutionary standpoint that class II hydrophobins have been found exclusively in Ascomycetes fungi and represent a uniform group in the phylogenic tree, while class I hydrophobins are found in both Basidiomycetes and Ascomycetes1,21. This lack of phylogenic uniformity and the obvious differences in structure and function between class I and class II hydrophobins prompted Whiteford and Spanu to speculate that class I and class II hydrophobins evolved independently and represent a case of convergent evolution21. In the present study, our structural and functional analysis of disulfide bonds in a class II hydrophobin have provided more evidence for the notion that class I and class II hydrophobins may have evolved independently, and suggest further research into the fundamental differences between class I and class II hydrophobins. Conclusions In this study, we found that the disulfide bridges of a recombinantly produced class II hydrophobin fusion protein were required for structure, surface activity at liquid-liquid and liquid-solid interfaces, and molecular self-recognition in solution multimerization. These findings are in agreement with previous results where disulfide bonds in the class II hydrophobin


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HFBI are required for functional separation in aqueous two phase systems using nonionic surfactants, and show that this relationship is robust in other interfacial contexts. The disulfide dependence of structure and function in class II hydrophobins is in contrast to what is known about class I hydrophobin disulfide bonds, which are dispensable for aggregation17,19, as well for formation of assembled structures on surfaces, and in part for protein solution structure17. Thus, while disulfides are largely conserved across class I and class II hydrophobins, the structural and functional consequences of these disulfides are different between class I and class II. Such differences in the disulfide roles could be the result of differences in the other structural elements between class I and class II. Supporting Information IAM/+DTT treated protein solubility assessment (Figure S1), IAM/-DTT versus IAM/+DTT protein PTFE binding (Figure S2) Present Addresses †Department of Chemical Engineering, University of Virginia, 214 Chem. Eng., 102 Engineers’ Way, Charlottesville, VA 22904 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding information This material is based upon work supported by the National Science Foundation under the CAREER program, Grant No. 1452855. Additional support for K.J.D was provided by a Grant for Experiential Learning in Health (GELH) from Lehigh University.


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ACKNOWLEDGMENTS We thank Dr. Ivan Korendovych of Syracuse University for providing Pichia pastoris strain GS115, and Dr. Olga Makhlynets for assistance with protein expression. REFERENCES (1) Linder, M. B., Szilvay, G. R., Nakari-Setälä, T., and Penttilä, M. E. (2005) Hydrophobins: the protein-amphiphiles of filamentous fungi. FEMS Microbiol. Rev. 29, 877–896. (2) Wessels, J. G. H. (1994) Developmental regulation of fungal cell wall formation. Annu. Rev. Phytopathol. 32, 413–437. (3) Wessels, J. G. H. (1996) Hydrophobins: Proteins that Change the Nature of the Fungal Surface, in Advances in Microbial Physiology (Poole, R. K., Ed.), pp 1–45. Academic Press. (4) Linder, M. B., Qiao, M., Laumen, F., Selber, K., Hyytiä, T., Nakari-Setälä, T., and Penttilä, M. E. (2004) Efficient Purification of Recombinant Proteins Using Hydrophobins as Tags in Surfactant-Based Two-Phase Systems. Biochemistry 43, 11873–11882. (5) Zampieri, F., Wösten, H. A. B., and Scholtmeijer, K. (2010) Creating Surface Properties Using a Palette of Hydrophobins. Materials (Basel). 3, 4607. (6) Bayry, J., Aimanianda, V., Guijarro, J. I., Sunde, M., and Latgé, J.-P. (2012) Hydrophobins— Unique Fungal Proteins. PLOS Pathog. 8, e1002700. (7) Niu, B., Wang, D., Yang, Y., Xu, H., and Qiao, M. (2012) Heterologous expression and characterization of the hydrophobin HFBI in Pichia pastoris and evaluation of its contribution to the food industry. Amino Acids 43, 763–771. (8) Linder, M. (2009) Hydrophobins: Proteins that self assemble at interfaces. Curr. Opin. Colloid Interface Sci. 14, 356–363. (9) Lo, V. C., Ren, Q., Pham, C. L. L., Morris, V. K., Kwan, A. H., and Sunde, M. (2014) Fungal Hydrophobin Proteins Produce Self-Assembling Protein Films with Diverse Structure and Chemical Stability. Nanomaterials 4, 827–843. (10) Hakanpää, J., Szilvay, G. R., Kaljunen, H., Maksimainen, M., Linder, M., and Rouvinen, J. (2006) Two crystal structures of Trichoderma reesei hydrophobin HFBI—The structure of a protein amphiphile with and without detergent interaction. Protein Sci. 15, 2129–2140. (11) Kallio, J., Paananen, A., Askolin, S., Nakari-Setälä, T., Parkkinen, T., Penttilä, M., Linder, M., and Rouvinen, J. (2004) Atomic Resolution Structure of the HFBII Hydrophobin, a Selfassembling Amphiphile. J. Biol. Chem. 279, 534-539. (12) Kisko, K., Szilvay, G. R., Vainio, U., Linder, M. B., and Serimaa, R. (2008) Interactions of


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Hydrophobin Proteins in Solution Studied by Small-Angle X-Ray Scattering. Biophys. J. 94, 198–206. (13) Grunér, M. S., Paananen, A., Szilvay, G. R., and Linder, M. B. (2017) The dynamics of multimer formation of the amphiphilic hydrophobin protein HFBII. Colloids Surfaces B Biointerfaces 155, 111–117. (14) Szilvay, G. R., Kisko, K., Serimaa, R., and Linder, M. B. (2007) The relation between solution association and surface activity of the hydrophobin HFBI from Trichoderma reesei. FEBS Lett. 581, 2721–2726. (15) Kisko, K., Szilvay, G. R., Vuorimaa, E., Lemmetyinen, H., Linder, M. B., Torkkeli, M., and Serimaa, R. (2009) Self-Assembled Films of Hydrophobin Proteins HFBI and HFBII Studied in Situ at the Air/Water Interface. Langmuir 25, 1612–1619. (16) Lienemann, M., Grunér, M. S., Paananen, A., Siika-aho, M., and Linder, M. B. (2015) Charge-Based Engineering of Hydrophobin HFBI: Effect on Interfacial Assembly and Interactions. Biomacromolecules 16, 1283–1292. (17) de Vocht, M. L., Reviakine, I., Wösten, H. A. B., Brisson, A., Wessels, J. G. H., and Robillard, G. T. (2000) Structural and functional role of the disulfide bridges in the hydrophobin SC3. J. Biol. Chem. 275, 28428–28432. (18) Talbot, N. J., Kershaw, M. J., Wakley, G. E., De Vries, O. M. H., Wessels, J. G. H., and Hamer, J. E. (1996) MPG1 Encodes a Fungal Hydrophobin Involved in Surface Interactions during Infection-Related Development of Magnaporthe grisea. Plant Cell 8, 985–999. (19) Kershaw, M. J., Thornton, C. R., Wakley, G. E., and Talbot, N. J. (2005) Four conserved intramolecular disulphide linkages are required for secretion and cell wall localization of a hydrophobin during fungal morphogenesis. Mol. Microbiol. 56, 117–125. (20) Linder, M., Selber, K., Nakari-Setälä, T., Qiao, M., Kula, M.-R., and Penttilä, M. (2001) The Hydrophobins HFBI and HFBII from Trichoderma reesei Showing Efficient Interactions with Nonionic Surfactants in Aqueous Two-Phase Systems. Biomacromolecules 2, 511–517. (21) Whiteford, J., and Spanu, P. (2002) Hydrophobins and the interactions between fungi and plants. Molecular Plant Pathology 3, 391-400. (22) Wu, S., and Letchworth, G. J. (2004) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 2004 v.36, 152– 154. (23) Schägger, H., and von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379. (24) Noguchi, T., Takahashi, C., Kimura, T., Muranishi, S., and Sezaki, H. (1975) Mechanism of the Intestinal Absorption of Drugs from Oil-in-Water Emulsions. VI. Absorption of LipidSoluble Dyes from Tributyrin and Triolein Emulsions in Rat Small Intestine. Chem. Pharm. Bull. (Tokyo). 23, 775–781.


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(25) Schneider, C. A., Rasband, W. S., and Eliceiri, K. W. (2012) NIH Image to ImageJ: 25 years of Image Analysis. Nat. Methods 9, 671–675. (26) Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D., and Bairoch, A. (2005) Protein identification and analysis tools on the ExPASy server. Humana, Totowa, NJ: pp. 571-607. (27) Lohrasbi-Nejad, A., Torkzadeh-Mahani, M., and Hosseinkhani, S. (2016) Heterologous expression of a hydrophobin HFB1 and evaluation of its contribution to producing stable foam. Protein Expr. Purif. 118, 25–30. (28) Kottmeier, K., Günther, T. J., Weber, J., Kurtz, S., Ostermann, K., Rödel, G., and Bley, T. (2012) Constitutive expression of hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris and its pre-purification by foam separation during cultivation. Eng. Life Sci. 12, 162–170. (29) Anson, M. L. (1940) THE REACTIONS OF IODINE AND IODOACETAMIDE WITH NATIVE EGG ALBUMIN. J. Gen. Physiol. 23, 321–331. (30) Askolin, S., Linder, M., Scholtmeijer, K., Tenkanen, M., Penttilä, M., de Vocht, M. L., and Wösten, H. A. B. (2006) Interaction and Comparison of a Class I Hydrophobin from Schizophyllum commune and Class II Hydrophobins from Trichoderma reesei. Biomacromolecules 7, 1295–1301. (31) Lakowicz, J. R. (1999) Energy Transfer, in Principles of Fluorescence Spectroscopy (Lakowicz, J. R., Ed.), pp 367–394. Springer US, Boston, MA. (32) Szilvay, G. R., Nakari-Setälä, T., and Linder, M. B. (2006) Behavior of Trichoderma reesei Hydrophobins in Solution: Interactions, Dynamics, and Multimer Formation. Biochemistry 45, 8590–8598. (33) Niu, B., Gong, Y., Gao, X., Xu, H., Qiao, M., and Li, W. (2014) The functional role of Cys3–Cys4 loop in hydrophobin HGFI. Amino Acids 46, 2615–2625.


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For Table of Contents Use Only


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A Structural and Functional Role for Disulfide Bonds in a Class II

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