Article pubs.acs.org/JPCB

Spontaneous Surface Self-Assembly in Protein−Surfactant Mixtures: Interactions between Hydrophobin and Ethoxylated Polysorbate Surfactants Ian M. Tucker,† Jordan T. Petkov,† Jeffrey Penfold,*,‡,§ Robert K. Thomas,§ Peixun Li,§ Andrew R. Cox,∥ Nick Hedges,∥ and John R. P. Webster‡ †

Unilever Research and Development Laboratory, Port Sunlight, Quarry Road East, Bebington, Wirral L63 3JW, United Kingdom ISIS, STFC, Rutherford Appleton Laboratory, Chilton, Didcot OX11 0QX, Oxfordshire, United Kingdom § Physical and Theoretical Chemistry Laboratory, Oxford University, South Parks Road, Oxford OX1 3QZ, Oxfordshire, United Kingdom ∥ Unilever Research Laboratories, Sharnbrook MK44 1LQ, Bedfordshire, United Kingdom ‡

S Supporting Information *

ABSTRACT: The synergistic interactions between certain ethoxylated polysorbate nonionic surfactants and the protein hydrophobin result in spontaneous self-assembly at the air−water interface to form layered surface structures. The surface structures are characterized using neutron reflectivity. The formation of the layered surface structures is promoted by the hydrophobic interaction between the polysorbate alkyl chain and the hydrophobic patch on the surface of the globular hydrophobin and the interaction between the ethoxylated sorbitan headgroup and hydrophilic regions of the protein. The range of the ethoxylated polysorbate concentrations over which the surface ordering occurs is a maximum for the more hydrophobic surfactant polyoxyethylene(8) sorbitan monostearate. The structures at the air− water interface are accompanied by a profound change in the wetting properties of the solution on hydrophobic substrates. In the absence of the polysorbate surfactant, hydrophobin wets a hydrophobic surface, whereas the hydrophobin/ethoxylated polysorbate mixtures where multilayer formation occurs result in a significant dewetting of hydrophobic surfaces. The spontaneous surface self-assembly for hydrophobin/ethoxylated polysorbate surfactant mixtures and the changes in surface wetting properties provide a different insight into protein−surfactant interactions and potential for manipulating surface and interfacial properties and protein surface behavior.

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INTRODUCTION

showed how intermolecular forces can be modified to produce molten protein phases by surface engineering of globular proteins using surfactant adsorption. Most proteins have a degree of surface activity and adsorb at a variety of different surfaces.5,8 Protein−surfactant adsorption has been explored for several systems,4,9 and the patterns of adsorption have some similarities with those encountered in some polyelectrolyte−surfactant mixtures.10 This is, for example, epitomized by the pattern of adsorption reported by Green et al.9 for lysozyme−sodium dodecyl sulfate, SDS, mixtures at the air−water interface. At low SDS concentrations there is a significant enhancement in the adsorption of the lysozyme and SDS because of surface complex formation. At higher SDS concentrations there is marked decrease in the complex adsorption as its solubility increases, and the surface is

The nature of protein−surfactant interactions is of central importance in many biomedical and biotechnological areas,1 such as in aspects of biocompatibility and biofunctionality. The role of ionic surfactants in promoting protein denaturation is well-established.1 In the particular context of this paper, the nature of protein−surfactant adsorption and interaction at surfaces is also of importance in its application in home and personal care products, cosmetics, pharmaceuticals, and food formulations.2−5 Nonionic surfactants generally interact more weakly with proteins than ionic surfactants and therefore tend not to cause denaturation. However, the different nature of their interaction provides other opportunities. Nonionic surfactants tend to solubilize proteins and suppress aggregation. Hence, their chaperoning ability can promote greater stability and the ethoxylate groups of the nonionic surfactants can be used to inhibit protein adsorption.6 The ability of surfactant adsorption to modify protein properties and self-assembly is illustrated by the recent work of Perriman and Mann.7 They © 2014 American Chemical Society

Received: March 10, 2014 Revised: April 15, 2014 Published: April 16, 2014 4867

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paper, where more complex surface structures are observed for hydrophobin/ethoxylated sorbitan surfactant mixtures. Related to the ethoxylated polysorbate surfactants used in this study are the more commonly encountered Tween nonionic surfactants. The Tween surfactants are ethoxylated sorbitan esters of different fatty acids. The series of surfactants Tween 20, 40, 60, and 80, all have 20 ethylene oxide groups attached to the sorbitan in the headgroup, which is attached to a single long-chain carboxylic acid, lauric, palmitic, stearic, and oleic respectively (see Figure 1b). They are an important class of food-grade surfactants, which are extensively used in emulsion and foam stabilization.6,20 In biomedical applications, where nonspecific protein adsorption can be undesirable, ethoxylate groups, and hence the Tween surfactants, are effective in the development of protein-resistant coatings and surfaces. The chaperoning qualities of ethoxylated nonionic surfactants, and in particular the Tween surfactants, have been investigated and exploited to suppress protein aggregation and to facilitate protein refolding in recombinant systems.21−23 Some specific binding mechanisms have been reported. For example, Zadymova et al.24 interpreted surface tension and light-scattering data in terms of complexation and association in Tween 80−bovine serum albumin (BSA) mixtures due to hydrogen bond formation between tryptophan residues on the BSA and the Tween ethylene oxide groups. In contrast, Kragel et al.25 reported the competitive adsorption between βlactoglobulin and β-casein with Tween 20, consistent with that reported for many other protein−surfactant mixtures.3,9,17 We report how the unique properties of both hydrophobin and some ethoxylated polysorbate nonionic surfactants combine to promote some unusual surface properties. The resultant surface structures are characterized by neutron reflectivity (NR), which in combination with H/D isotopic substitution reveals the complexity of the surface structures at the air−water interface arising from the hydrophobin/ ethoxylated polysorbate surfactant mixtures. The ethoxylated polysorbate surfactants used here were specifically synthesized with a lower degree of ethoxylation but are closely related in structure to the more commonly encountered Tween surfactants which do not show the observed surface ordering.

then dominated by the SDS adsorption. These adsorption trends correlate with the surface tension behavior9 and changes in the surface rheological properties.3 Gunning et al.11 have described a rather different behavior arising from quite different nonequilibrium conditions, where orogenic displacement of a spread protein layer at the air−water interface by a dilute surfactant solution occurs because of competitive adsorption. The unusual properties and pronounced surface activity of the protein hydrophobin have recently attracted considerable interest and attention.12,13 Hydrophobin is a small (∼10 kDa) globular protein which is produced by filamentous fungi. The structure of hydrophobin, with a central β-barrel structure and a small α-helix segment, is almost globular (see Figure 1a). The

Figure 1. Structure of (a) hydrophobin and (b) Tween 20 surfactant.

eight cysteine residues which form four intramolecular disulfide bridges make the protein both compact and rigid. The pronounced surface activity arises from a hydrophobic patch consisting of side-chain residues of leucine, valine, and analine, which occupy 20% of the surface area. The surface activity results in self-assembly at interfaces to form monolayer or bilayer structures, and these have been characterized by a variety of techniques.14−19 The strong adsorption and high elasticity of the adsorbed layers are important features for applications involving emulsion and foam stabilization.18,19 The adsorption and adherence to hydrophobic surfaces are also key to the biological function of hydrophobin and its application in surface coatings.12−16 Recently Zhang et al.17 have studied the adsorption of hydrophobin and hydrophobin/surfactant mixtures at the air− water interface using neutron reflectivity. The results show that hydrophobin adsorbs strongly to form a dense layer (volume fraction, φ ∼ 0.7) monolayer ∼30 Å thick, with a mean area/ molecule of ∼400 Å2 for concentrations >0.01 g/L. In competition with cationic, anionic, and nonionic surfactants, hexadecyltrimethylammonium bromide, CTAB, SDS, and hexaethylene glycol monododecyl ether, C12E6, the hydrophobin dominates the adsorption for surfactant concentrations below the surfactant critical micellar concentration (CMC). Above the CMC of the surfactants, the hydrophobin is displaced at the surface by the surfactant. For C12E6, the displacement is only partial. Some hydrophobin remains at the surface above the surfactant CMC and is indicative of a surface interaction between the hydrophobin and C12E6. At pH 3 the pattern of adsorption for SDS−hydrophobin and C12E6− hydrophobin mixtures is more complex in the region of the CMC, and a layered structure is formed at the interface. These two latter observations provide a direct link to the focus of this

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EXPERIMENTAL DETAILS

The NR measurements were made on the INTER reflectometer26 at the ISIS pulsed neutron source at Rutherford Appleton Laboratory, U.K. The reflectivity R(Q) was recorded using a single detector at a fixed grazing angle of incidence θ of 2.3° and a neutron wavelength λ ranging from 0.5 to 15 Å to provide measurements of R(Q) over a wave vector transfer Q (Q = 4π sin θ/λ) range of 0.03−0.5 Å−1. The absolute values of the reflectivity were calibrated with respect to the direct beam and the reflectivity from a D2O surface. The measurements were made in sealed Teflon troughs at 25 °C using ∼25 mL sample volumes. Each neutron reflectivity profile was measured for ∼30 min. The measurements were made sequentially on a seven-position sample changer. They were repeated at least once until the reflectivity showed no significant variation with time, to give a total lapse time of ∼3 to 6 h. In the kinematic approximation, the specular reflectivity is related to the square of the Fourier transform of the scattering length density profile ρ(z), normal to the interface27 4868

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16π 2 Q2

∫ ρ(z)e−iQz dz

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multilayer formation and rinsing. A 20 μL drop of water were used to create a droplet on the surface of a treated silicon wafer substrate. The silicon surfaces were oxidized and hydrophobized using a sulfosilazine agent and exposed to solutions which exhibited either monolayer or multilayer adsorption. The images were captured using the manufacturer’s recommended setting and adjusted for optimal focus. The contact angle, where possible, was evaluated from static images using the circle algorithm and a manual background. The video images were captured at a rate of 25 frames/second for a total of 100 frames. The NR measurements were made for PES8-20, 40, 60, and 80 in nrw, and hydrophobin/PES8-20 (40, 60, and 80) mixtures in nrw. The values of the sum of scattering lengths, ∑b, of the different materials used in this study are summarized in Table S1 in the Supporting Information.

2

(1)

where ρ(z) = ∑ini(z)bi, ni(z) is the number density of ith nucleus, and bi the scattering length. In the NR measurements, ρ(z) can be manipulated by using hydrogen (H)/deuterium (D) isotopic substitution (H and D have different neutron scattering lengths, −3.71 × 10−5 Å for H and 6.674 × 10−5 Å for D), and this manipulation of the “contrast” has been extensively exploited at the air−water interface for the study of the adsorption of a range of surfactants and polymer−surfactant mixtures.27,28 The NR measurements reported here are all made in null reflecting water (nrw; 92 mol % H2O, 8 mol % D2O mixture with a scattering length of zero, the same as that of air) at 25 °C. The reflected signal then arises only from any surface layer with a scattering length density different from zero. The NR data reported here correspond to surface structures that range from a simple monolayer to a more complex layered structure comprising up to six layers (see following text for details). The specular reflectivity can be equally well-described using the kinematic approximation (eq 1) or the more exact optical matrix method.27 The reflectivity data here are fitted by comparing them with a profile calculated using the optical matrix method for the simplest structural model that is consistent (on a least-squares evaluation) with the data and is physically consistent with the dimensions and densities of the different components. Each layer in the model is characterized by a thickness d and a scattering length density ρ. In the modeling of the NR data, the simplest model, that is, the one with the least number of layers, consistent with the data is used. The hydrophobin, class II hydrophobin, HFBII, was produced by yeast fermentation at an external fermentation company (BAC) and purified by a two-phase extraction, as described elsewhere.17 The extracted and purified material was freeze-dried before preparation of the aqueous solutions. The solutions were prepared in degassed water to minimize bubble formation. Four different ethoxylated polysorbate surfactants were synthesized in Oxford29 by ethoxylation (using deuteriumlabeled ethylene oxide) of the appropriate sorbitan ester,30 which was supplied by Sigma-Aldrich. The polyethylene(8) sorbitan monolaurate, monopalmitate, monostearate, and monooleate surfactants were prepared with eight deuteriumlabeled ethylene oxide groups. This contrasts with the commonly used Tween surfactants, which have 20 ethylene oxide groups. For consistency with the Tween nomenclature, we abbreviate the surfactants synthesized here as PES8-20, PES8-40, PES8-60, and PES8-80. The surface purity of the ethoxylated polysorbate surfactants was verified from the NR measurements. High-purity water (Elga Ultrapure) was used, and the D2O was obtained from Sigma-Aldrich. All the glassware and Teflon troughs (used for the NR measurements) were cleaned in alkali detergent (Decon 90) and rinsed thoroughly in high-purity water. All the solutions were carefully degassed using mild ultrasound, as in the previously reported surface and solution studies on hydrophobin/surfactant mixtures.17,31 This avoids the self-assembly into large aggregates reported in some studies. 12 All the solutions were clear, and routine DLS measurements showed no evidence of large aggregates. Contact angle measurements were made using a Kruss DSA100 drop shape analyzer to quantify the changes in the wetting properties of a hydrophobic surface after surface

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RESULTS AND DISCUSSION Adsorption of Ethoxylated Polysorbate Surfactants at the Air−Solution Interface. The adsorption properties of the PES8-20, 40, 60, and 80 were initially characterized at the air− water interface using NR to establish the adsorption isotherms in the absence of hydrophobin. The deuterium-labeled ethylene oxide groups of the ethoxylated sorbitan headgroup provided the required “contrast” to observe and evaluate the adsorption. The resulting adsorption isotherms are shown in Figure 2.

Figure 2. Adsorption isotherms for (red) PES8-20, (blue) PES8-40, (green) PES8-60, and PES8-80 (black).

The measurements were made in nrw, and the reflectivity was evaluated as a single monolayer, with d and ρ values as summarized in Table S2 in the Supporting Information. The area/molecule and adsorbed amount are evaluated in the usual way27 using

A=

∑b dρ

(2)

where the Σb values are listed in Table S1 in the Supporting Information. The adsorption isotherms are consistent with a CMC ∼ 10 to 20 μM, which is similar to that reported for the Tween surfactants.30 For all four surfactants, the adsorbed layer is ∼20 ± 1 Å in thickness and the saturation adsorption corresponds to a mean area/molecule of ∼65 Å2 and an adsorbed amount of Γ∼ 2.6 × 10−10 mol cm−2. The saturation 4869

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length ρ; the reflectivity is calculated exactly using the familiar optical matrix formulism from thin-film optics and described in detail elsewhere.27 In Figure 3, at the highest surfactant concentration (1000 μM), the NR data are from a monolayer of ∼20 Å, consistent with a surface dominated by ethoxylated polysorbate surfactant adsorption. Assuming only surfactant at the interface, this corresponds to an area/molecule of ∼77 Å2, compared to ∼65 Å2 for a saturated layer (see Figure 2 and Table S2 in the Supporting Information). At the lowest surfactant concentration (3 μM), the NR data are also from a monolayerof ∼30 Å. This is consistent with a surface dominated by the hydrophobin adsorption, with an area/molecule of ∼445 Å2, close to the saturated value of ∼420 Å2.17 These results are consistent with the previously reported observations of competitive adsorption between hydrophobin and a range of different ionic and nonionic surfactants.17 The NR data in Figure 3 and the model parameters in Table 1 show that for PES8-60 concentrations between 550 and 6.25 μM the surface structure is more complex than a single monolayer. It corresponds to a thicker layer. However, the reflectivity is not consistent with a single layer of uniform density, and can be described only by a layered structure in which the refractive index varies. The NR data comprises two principle interference features which are most developed at the intermediate surfactant concentrations. The data where the surface structure is most well-developed are best modeled by a layered structure at the interface as depicted approximately in the schematic diagram in Figure 4 and as established from the model parameters in Table 1. For PES8-60, the structure is best described as an initial monolayer (∼15−20 Å) of deuterated ethoxylated polysorbate surfactant followed by layers of hydrophobin (∼30 Å) separated by thin layers (∼10−15 Å) that contain principally the deuterated ethoxylated polysorbate headgroups. At the extremes of the surfactant concentration range this structures is less well-defined and developed and can be described by a smaller number of layers. Although there is some variation in the structural parameters, the general model obtained and illustrated in Figure 4 is similar for all the mixtures measured. If the adjacent ethoxylated− hydrophobin layers are treated as a pair, then the structure is basically in the form of 3 bilayers. The initial interference fringe in the NR data at low Q values (Q ∼ 0.08 Å−1) relates to the total layer thickness. The second interference fringe at higher Q values (Q ∼ 0.15 Å−1) is a broad Bragg peak, which is broadened because of the limited number of bilayers at the interface. Repeated measurements indicate that the formation of the layered structures at the interface occurred relatively quickly and that there was no marked long-term evolution in the surface structure (on the time scale of the measurements, which was typically ∼3−6 h; see Experimental Details for more details of the measurement regime). Under the range of conditions explored the structure never required more than a maximum of six layers (three bilayers) at the interface, and often less were necessary. The modeling of the NR data is highly sensitive to the structure of the adsorbed layer because of the dimensions involved and because the measurements in nrw with the ethylene oxide groups of the ethoxylated polysorbate surfactant headgroup deuterium labeled provides an advantageous contrast profile, or refractive index distribution (see Table S1 of the Supporting Information for the respective ∑b values).

adsorption is broadly similar to that previously reported for octaethylene monododecyl ether, C12E8.27 The values for the individual surfactants with surfactant concentration are summarized in Table S2 in the Supporting Information. The similarity of the adsorption for the four surfactants indicates that the headgroup geometry dominates the surface packing and adsorption. However, in detail, the adsorption does increase slightly as the alkyl chain increases from laurate (PES8-20) to palmitate (PES8-40) and stearate (PES8-60). The unsaturated oleate (PES8-80) surfactant has a slightly larger area/molecule, comparable to that for PES8−20, because of the less favorable packing of the unsaturated alkyl chain. The adsorption isotherms for the different surfactants indicate that the slope in the adsorption below the CMC is increasingly steeper as the alkyl chain length increases. This is consistent with an increase in the cooperativity in the adsorption because of the greater hydrophobic attraction between the alkyl chains in the adsorbed layer. Adsorption of Hydrophobin/Ethoxylated Polysorbate Mixtures. A series of NR measurements were made in nrw for the combination of hydrophobin/ethoxylated polysorbate for PES8-20, 40, 60 and 80, at surfactant concentrations from ∼2 to 1500 μM and at two hydrophobin concentrations, 0.05 and 0.2 mg/mL. The NR data in Figure 3 show the evolution of the reflectivity for PES8-60/0.05 mg/mL hydrophobin as the surfactant concentration varies from 3 to 1000 μM.

Figure 3. NR data for PES8-60/0.05 mg/mL hydrophobin in nrw: (from bottom to top) (black) 1000 μM PES8-60, (red) 500, (blue) 275, (green) 165, (pink) 100, (cyan) 50, (yellow) 25, (gray) 12.5, (dark blue) 6.25, and (dark gray) 3.1. The solid lines are model calculations for the key model parameters summarized in Table 1. The different curves are shifted vertically for clarity.

A broadly similar evolution in the reflectivity and the surface structure occurs for PES8-20, 40, and 80 at both hydrophobin concentrations. The key model parameters for all the data are summarized in Tables S3−S6 in the Supporting Information, and those for the data in Figure 3 are summarized in Table 1. The approach to the modeling of the data, as described briefly in the Experimental Details, is to use the simplest model, the minimum number of layers, that adequately describes the data and is consistent with dimensions and scattering lengths of the different components. In the data presented here, the number of layers in the model varies from one to six. In detail, each layer is characterized by a thickness d and a scattering 4870

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Table 1. Key Model Parameters from the Analysis of the NR Data for PES8-60/0.05 mg/mL Hydrophobin PES8-60 conc (μM)

d1 (±2 Å)

ρ1 (±0.2 × 10−6 Å−2)

d2 (Å)

ρ2 (×10−6 Å−2)

d3 (Å)

ρ3 (×10−6 Å−2)

d4 (Å)

ρ4 (×10−6 Å−2)

d5 (Å)

ρ5 (×10−6 Å−2)

d6 (Å)

ρ6 (×10−6 Å−2)

1000 550 275 165 100 50 25 12.5 6.25 3.13

19 16 14 14 15 15 16 22 30 28

2.4 2.2 1.3 1.3 2.0 1.7 2.0 1.5 1.4 1.3

− 37 25 29 27 20 29 24 70 −

− 0.3 1.0 1.0 0.7 0.6 0.9 0.6 0.8 −

− 10 12 10 9 13 5 7 6 −

− 2.2 2.3 3.4 2.2 2.4 4.0 1.8 0.9 −

− 25 37 32 30 29 31 24 48 −

− 0.9 0.7 0.4 0.5 0.7 1.0 1.5 0.3 −

− 11 9 8 16 11 11 11 − −

− 2.8 3.7 3.8 2.0 3.2 1.9 1.9 − −

− 50 43 41 74 40 32 32 − −

− 0.5 0.9 0.9 0.5 0.6 0.5 0.5 − −

Figure 4. Schematic representation of the hydrophobin/ethoxylated polysorbate surfactant surface structure for the concentration regime where the structure comprises three bilayers. The thicknesses quoted are the average values from Table1 for surfactant concentrations from 12.5 to 550 μM.

Some limited measurements were made with a different contrast by making the measurements in D2O rather than in nrw. The data, model calculations, and key model parameters for 165 μM PES8-60/0.05 mg/mL hydrophobin and 50 μM PES8-60/0.05 mg/mL hydrophobin in nrw and in D2O are shown and summarized in Figure 5 and Table S7 in the Supporting Information. The data for the two different contrasts are consistent with the same structural model in which only the contrast of the solvent and solvent-containing layers is changed. Complementary data for PES8-20, 40, and 80 (see Tables S3−S6 in the Supporting Information) show that the surface structures are broadly similar but less well-developed and occur over a narrower surfactant concentration range than those for PES8-60. Hence, the structure of PES8-60 and the nature of the interaction between PES8-60 and hydrophobin appears optimal for the formation of these complex surface structures. Furthermore, the layered surface structures are also less welldeveloped at the higher hydrophobin concentrations of 0.2 mg/ mL compared to the structures formed at 0.05 mg/mL, as illustrated in Figure S1 and Tables S3−S6 in the Supporting Information. Indeed, for PES8-20/0.2 mg/mL hydrophobin, only monolayer adsorption is observed over the entire PES8-20 concentration range probed. The evolution in the surface structure for the hydrophobin/ ethoxylated polysorbate surfactant mixtures is represented by an approximate surface phase diagram, as shown in Figure 6 for 0.05 mg/mL hydrophobin. The complementary phase diagram

Figure 5. (a) NR data for 165 μM PES8-60/0.05 mg/mL hydrophobin in nrw (red) and in D2O (blue); (b) NR data for 50 μM PES8-60/0.05 mg/mL hydrophobin in nrw (red) and in D2O (blue).The solid lines are model calculations for the key model parameters summarized in Table S7 of the Supporting Information. The initial interference fringe at Q ∼ 0.08 Å−1 arises from the total film thickness, and the fringe at Q ∼ 0.15 Å−1 results from the bilayer structure and is a broadened Bragg peak as described in the main text.

for 0.2 mg/mL hydrophobin is shown in Figure S1 in the Supporting Information. Wetting Properties. The hydrophobin/ethoxylated polysorbate surfactant mixtures exhibit some interesting and unusual wetting properties on hydrophobic surfaces, such as Teflon. This is illustrated qualitatively on Teflon in the 4871

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Figure 6. Surface phase diagram for 0.05 mg/mL and PES8-20, 40, 60, and 80. See legend for further details.

photographs shown in Figure S2 in the Supporting Information. Solutions of hydrophobin and hydrophobin/ ethoxylated polysorbate surfactant mixtures, where only monolayer formation occurs at the air−water interface, strongly wet hydrophobic surfaces such as Teflon. The hydrophobin adsorption at the hydrophobic surface is irreversible and is not removed by simple rinsing. In some related NR measurements of hydrophobin onto an OTS surface32 it was shown that hydrophobin adsorbed irreversibly as a monolayer because of the strong interaction between the hydrophobic patch on the hydrophobin and the hydrophobic surface and rendered the surface hydrophilic. When exposed here to hydrophobin/ ethoxylated polysorbate surfactant solutions, where there is a layered structure at the air−water interface, the surface of Teflon remains highly hydrophobic. This indicates that the hydrophobin/ethoxylated polysorbate surfactant complexes do not wet the Teflon surface; hence, such surface assemblies must have a hydrophilic nature greater than that of hydrophobin alone. This is illustrated more quantitatively in Figure 7 in which the profile of a water droplet on a hydrophobized silicon surface is shown after exposure to monolayer adsorption of hydrophobin and rinsing (Figure 7a) and for a surface subject to hydrophobin/ethoxylated sorbitan solution that results in multilayer adsorption at the air−water interface (Figure 7b). This implies that there is a lack of hydrophobin adsorption at the hydrophobic solid surface and that hydrophobin is then preferentially incorporated into the multilayer structures. Consistent with the more qualitative observations in Figure S2 in the Supporting Information, the surface in Figure 7a is highly hydrophilic and wetting. The applied water droplet forms a thin wetting layer with a contact angle close to zero. This is also well-illustrated in the video clip available as Supporting Information. However, the surface exposed to the hydrophobin/ethoxylated polysorbate layered structure is now highly hydrophobic and the contact angle in Figure 7b is ∼90°. The wetting behavior described here is in marked contrast to the wetting behavior encountered in polyelectrolyte/ionic surfactant mixtures, as described for polyethylenimine (PEI)/ SDS mixtures.33 In that case, the strong surface complexation and surface multilayer formation between the PEI and SDS result in solutions which strongly, persistently, and irreversibly wet Teflon. In contrast, surfactant solutions at similar surfactant concentrations in the absence of PEI do not or only weakly wet Teflon. Discussion. Surface ordering has been previously reported in polyelectrolyte−ionic surfactant mixtures28 and for anionic surfactants with the addition of multivalent counterions, such as Ca2+33 and Al3+.34 An overriding feature of such surface

Figure 7. Water droplet profiles on a hydrophobized silicon surface after exposure and rinsing for (a) monolayer hydrophobin adsorption and (b) ethoxylated polysorbate/hydrophobin multilayer adsorption.

ordering is the strong electrostatic attraction and complexation between the polyelectrolyte or multivalent counterions and the ionic surfactants. In such systems the surface structure depends upon the detailed structure of the components and relative concentrations and can be tuned to form a range of structures that includes single bilayers and multilayers. The solutions that form these surface structures also show some remarkable wetting properties: they persistently wet hydrophobic surfaces such as Teflon. This is in marked contrast to the wetting behavior of the ethoxylated polysorbate surfactant−hydrophobin mixtures discussed above. The hydrophobin−surfactant mixtures previously studied at the air−water interface showed predominantly competitive adsorption.17 That is, either the hydrophobin or the surfactant dominated the surface depending upon the individual concentrations relative to the surfactant CMC. However, at pH 3, where the hydrophobin is weakly cationic, for a limited range of concentrations for hydrophobin/ SDS and hydrophobin/C12E6 in the region of the CMC, surface layering in the form of a single bilayer at the surface was 4872

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observed.17 The origin of the interaction associated with the ordering observed for these hydrophobin/surfactant mixtures was again primarily electrostatic because of the interaction between the SDS and C12E6 headgroup and hydrophilic regions of the hydrophobin. The surface structures obtained here for the hydrophobin/ ethoxylated polysorbate surfactant mixtures and their wetting properties suggest that the nature of the interaction is different for this mixture and is not dominated by an electrostatic interaction. The structure represented in Figure 4 is deduced from the structural data, with the assumption that the predominant interactions are (i) the hydrophobic interaction between the surfactant alkyl chain and the hydrophobic patch on the surface of the hydrophobin and (ii) the interaction between the ethoxylated headgroup of the surfactant and the hydrophilic region of the hydrophobin. Hence, as illustrated in Figure 4, the structure is best described as a “bricks and mortar” structure, where the hydrophobin represents the “bricks”, the surfactant headgroups the “mortar” between the horizontal layers, and the surfactant alkyl chains the “mortar” in the vertical gaps. The layers of hydrophobin could also be considered as layers of hydrophobin dimers. In terms of the multilayer formation, the hydrophobic interaction is optimal for the stearate alkyl chain length, with a fully extended chain length of ∼22 Å similar to the dimensions of the hydrophobic patch on the hydrophobin. But this does not necessarily imply that the chains are in a fully extended configuration in the surface layers. They are merely illustrated as such in Figure 4 for simplicity. The interaction appears less optimal for the shorter lauryl and palmitic alkyl chain lengths of PES8-20 and PES8-40 (∼17 and 20 Å, respectively). It is also less optimal for the unsaturated oleic alkyl chain length of PES8-80, presumably in that case as a result of less favorable packing of the alkyl chain. This is illustrated in Figure 6 and Figure S1 in the Supporting Information, which show the extent of the formation of the layered structures with surfactant concentration for the different surfactant alkyl chain geometries. The specific nature of the hydrophobic interaction between the ethoxylated polysorbate surfactants and hydrophobin and their relative concentrations on the surface layering is highlighted in this paper. However, two aspects of that further study are worthy of discussion here as they further highlight the sensitive nature of the interactions and structures which give rise to the surface ordering. Measurements with the Tween 60 surfactants (ethoxylated polysorbate surfactants with a degree of ethoxylation of 20) with hydrophobin do not show any evidence of surface ordering. The importance of the specific structure and packing associated with the ethoxylated polysorbate surfactants is illustrated in the absence of ordering in decaethylene monostearyl ether (C18E10)/hydrophobin mixtures. The particular structure and size of the hydrophobin protein is also important, and the coadsorption of PESn-60 with a range of other proteins with quite different structures, βlactoglobulin, β-casein, and lysozyme, showed no evidence of surface ordering. Although the basic structure, described as 3 bilayers of alternating layers of ethoxylated polysorbate surfactant and hydrophobin dimers, is robust over a range of surfactant and hydrophobin concentrations and for the different ethoxylated polysorbate surfactants, there are some variations. These variations in part reflect the changes in the interaction between the different surfactants and hydrophobin. In particular, the outermost hydrophobin layer, the layer adjacent to the solvent

phase, is often thicker than a single monolayer of dimers (see parameters in Table 1). Furthermore, at the extremes of the surfactant concentration range over which surface multilayer structures form, the surface structures are often less welldeveloped and have correspondingly thicker layers and a smaller number of layers in the structure. These could be associated with hydrophobin tetramer formation within the surface region rather than dimer formation at the interface. Dimers and tetramers are known to form in solution in the absence of surfactant,31,36 and more complex hierarchical structures have been invoked at surfaces.37 The greatest variations in the structure and the more disordered structures occur at the lower surfactant concentrations and higher hydrophobin concentration and for the two ethoxylated polysorbate surfactants with the shorter alkyl chain lengths, PES8-20 and PES8-40. This highlights the relative importance of the surfactant/protein concentration ratio and the hydrophobic interaction in determining the surface structure. In the regions where the surface structure is less well-defined and in general in the outermost layer (adjacent to the solvent phase), there could well be lateral inhomogeneities and other variations in the structure. Such inhomogeneities and variations could be associated with slow kinetics of reorganization at the surface, although as described in the Experimental Details none were observed on the time scale of the measurements. The outermost layers and the less well-developed structures could also be relatively dilute; hence, the structures will start to appear to be less distinct. The differences in the wetting properties of the hydrophobin/ethoxylated polysorbate surfactant mixtures for monolayer and multilayer formation are also consistent with a predominantly hydrophobic interaction between the hydrophobin and ethoxylated polysorbate surfactant. In the other systems where the surface multilayer formation is dominated by a strong electrostatic attraction28,31,34,35 the solutions which exhibit surface multilayer formation strongly wet hydrophobic surfaces like Teflon. In contrast, for surfactant monolayer adsorption at these concentrations, surfaces like Teflon are only weakly wetted or not at all. The wetting behavior of the hydrophobin/ethoxylated polysorbate surfactant mixtures is exactly opposite to this. For monolayer adsorption, as discussed earlier, the solutions wet Teflon because of the strong hydrophobic interaction between the Teflon surface and the hydrophobin. Whereas for the mixtures where surface multilayer formation occurs, the solutions do not wet Teflon, and this implies that the ethoxylated polysorbate surfactant− hydrophobin structures are predominantly hydrophilic. Finally, the results offer a different perspective on the nature of nonionic surfactant−protein interactions. Poly(ethylene oxide) chains are extensively used to inhibit the often undesirable nonspecific adsorption of proteins on surfaces, and in this role the related Tween surfactants have been extensively studied and exploited.6 Detailed studies on the interaction of the Tween surfactants with different proteins have been reported.20−25 While the competitive adsorption of Tween with different proteins, such as Soy Globulins20 and BSA,24 has been observed, Zadymova et al.24 also reported a specific hydrogen bonding interaction between Tween 80 and BSA. Bam et al.21,22 described the possible role of the Tween surfactants in acting as chaperones in the folding of recombinant human growth hormone by binding to the hydrophobic portions of the native protein. It is generally assumed that the polyoxyethylene-based nonionic surfactants 4873

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have a lower tendency to induce protein denaturation. Le Marie et al.38 and Arachea et al.39 have discussed the role of nonionic surfactants, and in particular the Tween surfactants, in membrane protein extraction, solubilization, and stabilization. It is evident that the Tween surfactants can be highly efficient for the extraction of some proteins and the least efficient for others. Here we have reported a new aspect of the binding of nonionic surfactants with proteins. The structure of the ethoxylated polysorbate surfactant, with a degree of ethoxylation lower than that of the commonly used and related Tween surfactants, and that of the hydrophobin provide the opportunity for hydrophobic and hydrophilic interactions between the surfactant and the protein. This provides the optimal criteria for the formation of the unusual surface multilayer structures reported here, which have not been reported for any other surfactant/protein mixtures. In addition, the range of ethoxylated polysorbate surfactants available from the different alkyl chain lengths offers the potential for the manipulation of the protein−surfactant interaction over a wider range of potential applications. This could impact the chaperoning function in protein folding,21,22 stabilization of protein aggregates, modification of protein properties and selfassembly,7 and efficient delivery and control of proteins at interfaces. As such they have the potential to impact home and personal care products, cosmetics, pharmaceuticals, food formulations and biomedical/biotechnological applications.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the provision of beam time on the INTER reflectometer at the ISIS neutron source and the invaluable assistance of the instrument scientists and technical support.

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CONCLUSIONS The ethoxylated polysorbate surfactant−hydrophobin mixtures exhibit some remarkable surface adsorption properties, in the form of well-defined layered surface structures. The formation of these structures is promoted by specific interactions between the surfactant and hydrophobin, arising from the interaction of the surfactant alkyl chain with the hydrophobic patch on the surface of the hydrophobin and the interaction between the ethoxylated headgroup of the surfactant and hydrophilic regions of the hydrophobin. In particular, the way the variation in the hydrophobic interaction and packing varies with the alkyl chain length of the ethoxylated polysorbate surfactant is highlighted. The ethoxylated polysorbate−hydrophobin solutions exhibit contrasting wetting properties on hydrophobic surfaces, depending upon the nature of the surface adsorption. The surface monolayers wet hydrophobic surfaces, whereas the surface multilayer structures do not. This is driven by the predominantly hydrophobic interaction between ethoxylated polysorbate surfactant and hydrophobin. It also contrasts with the wetting behavior of surface multilayers formed by a strong electrostatic interaction, such as the polyelectrolyte/surfactant mixtures,28 where the opposite wetting behavior exists. The spontaneous surface self-assembly and unique wetting properties provide new opportunities for manipulating surface and interfacial properties and protein behavior.

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ASSOCIATED CONTENT

S Supporting Information *

Additional tables of model parameters, NR data, and a surface phase diagram; a video clip, in .AVI format, of the surface wetting properties. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: jeff[email protected]. 4874

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