Altering Hydrophobin Film with Ethanol

Controlling the Self Assembly of Hydrophobin Films

  • Tab. 1: Surface tension relation to ethanol: water ratio [9]Tab. 1: Surface tension relation to ethanol: water ratio [9]
  • Tab. 1: Surface tension relation to ethanol: water ratio [9]
  • Fig. 1: AFM topographical images of hydrophobin films self-assembled on HOPG from aqueous droplets containing different amounts of ethanol. A) Hydrophobin film formed from MQW. The hydrophobin film is comprised of laterally packed rodlets, with length ranging between 50 nm and 200 nm, which self-assemble into a tightly packed protein film. Average height of the surface is ~2.6 nm. B) Hydrophobin film formed from 5% ethanol. Addition of ethanol induces the hydrophobins to form longer rodlets, typically 100 to 400 nm long, suggesting that a lower surface tension may stabilize the hydrophobins at the surface of the droplet. C) Hydrophobin film formed from 15% ethanol. At this concentration, ethanol results in changes to the lateral packing of the rodlets, resulting in triangular/parallelogram shaped holes throughout the surface of the film. The diameter of these holes ranges from 18 nm to 300 nm. D) Hydrophobin film formed from 25% ethanol contains large sections of parallel-packed rodlets. The measured average height of the film prepared under these conditions is 4.2 nm, indicating a change in molecular packing of the hydrophobin.
  • Fig. 2: Water contact angle on bare HOPG and on hydrophobin film-coated HOPG surfaces, with films formed from protein solutions containing a range of ethanol concentrations. Coating of HOPG with a monolayer of EASΔ15 protein reduces the contact angle of water on HOPG from ~85° (bare HOPG) to ~50° (film formed from 25% ethanol). Error bars and values represent standard error (SEM).

Hydrophobins are fungal proteins with many potential applications due to their ability to self-assemble into amphipathic films at hydrophobic:hydrophillic interfaces. The ability to manipulate surface characteristics of hydrophobin films is highly desirable. Here, we have shown that by adjusting the amount of ethanol in an aqueous solution containing the hydrophobin EASΔ15, protein films with distinct features and properties can be obtained, as confirmed by AFM and contact angle measurements.

Introduction to Hydrophobin Proteins

In recent years hydrophobin proteins have received significant research attention due their unique properties and potential for use in a wide variety of applications, such as in drug delivery, food preservation and the preparation of anti-icing and anti-biofouling coatings [1]. Hydrophobins are a family of small proteins found only in the fungal kingdom. They are secreted by filamentous fungi to fulfil different roles during growth and development. The function of hydrophobin proteins can vary, from acting as surfactants that lower the surface tension of water, to forming protective coatings around spores that prevent immune responses in mammals, stabilizing structures around air channels, recruiting cutinases prior to germination, repelling water and acting as an adhesive surface [2,3]. At hydrophobic:hydrophilic interfaces, hydrophobin proteins can
self-assemble into amphipathic films composed of rod-like motifs, rich in β-sheet structure, which are called rodlets. These hydrophobin rodlet films can alter the wettability of surfaces and have been shown to be extremely resilient compared to other protein films, being able to withstand high temperatures and pressure, while being chemically stable under both acidic and basic conditions [4].  These properties make hydrophobin films extremely appealing for coating applications. However, to exploit and manipulate the unique nature of these proteins to their full potential, better control of the self-assembly process is required.

Coating Graphite with a Hydrophobin Film

The hydrophobin protein EASΔ15 was used to investigate the effects of surface tension on the self-assembly of hydrophobin films from solution.

Ethanol was used to prepare a series of solutions with a range of ethanol concentration and surface tension (tab. 1). Lyophilized EASΔ15 was dissolved in Milli-Q water (MQW) or MQW containing 5%, 15% or 25% v/v ethanol at a protein concentration of
5 µg/ml. A 50-µl droplet of the EASΔ15 protein solution was incubated highly oriented pyrolytic graphite (HOPG) surface overnight to allow self-assembly into a film. The films were rinsed under a stream of MQW for 30 seconds to remove excess protein and then dried at 70°C to evaporate the ethanol and water.

AFM Investigations of Hydrophobin Surface

The surfaces of the EASΔ15 films produced from solutions of different ethanol concentration and surface tension were analyzed with atomic force microscopy in tapping mode. For each of the EASΔ15 films, three different regions were examined. AFM of the EASΔ15 film produced from MQW reveals closely packed rodlet structures, with rodlet structures and dimensions corresponding to a monolayer (~2.6 nm) and consistent with reports in the literature [2,4,5] (fig. 1A). The film produced from 5% ethanol exhibits an altered morphology with longer rodlets ranging from 50–200 nm and average height of ~2.7 nm (fig. 1B). As the concentration of ethanol is increased, the structure of the EASΔ15 films formed changes. Films formed from 15% ethanol display more obvious and larger holes throughout, with triangular or parallelogram shapes evident (fig. 1C). At the highest concentration of ethanol used, 25%, EASΔ15 self-assembles into a film with fewer holes and with large areas covered with long parallel rodlets (fig. 1D). The measured average height of the film in this case is 4.2 nm. These data indicate that the presence of ethanol as additive alters the mechanism of self-assembly of EASΔ15 or the intermolecular arrangement of the protein in the rodlets.

Water Contact Angle (WCA) on Hydrophobin Film

WCA is commonly used to investigate the ability of hydrophobin films to reverse surface wettability [2,6,7]. Here, a 10-µl droplet of MQW was placed on the surface of uncoated or hydrophobin-coated HOPG, allowing the droplet to rest for 5 minutes with a reading taken every minute and repeated in three different areas. Contact angles for each of the images were measured in ImageJ, using Dropsnake contact angle analysis program [8] and the average is reported (fig. 2). There is an overall reduction of the water contact angles with the hydrophobin coating. Water on the bare HOPG surface displays a contact angle of ~85°, and with a coating of EASΔ15 hydrophobin produced from MQW, this value is reduced to ~77° indicating an increase in wettability. The contact angle is further reduced by the hydrophobin films formed from solutions containing 5% and 15% ethanol, to 61° and 59°, respectively. For the hydrophobin film self-assembled from 25% ethanol, the water contact angle falls to 49°. Hydrophobin proteins are known to self-assemble to form amphipathic monolayers at hydrophobic:hydrophilic interfaces. Here, it appears that as the protein solutions evaporate, the hydrophobin films form on the HOPG and generate a hydrophilic coating. At higher ethanol concentrations, ethanol affects protein self-assembly and film morphology and properties. This may be due to its absorption to the solution surface, the HOPG surface or even to the hydrophobin surface, effectively altering the surface activity of the protein.


The nature of an interface is known to play an important role in the formation of hydrophobin films and without a distinct hydrophobic:hydrophilic interface hydrophobin rodlets do not form [2,4]. Reduction in surface tension by addition of ethanol is known to affect the rate of self-assembly of hydrophobins in solution [6]. Here we have shown that the addition of ethanol can be used to control self-assembly of the hydrophobin EASΔ15 onto a solid surface from solution and that morphology and properties of the protein film can be tuned, including thickness, porosity and wettability. Future research is required to probe the behavior of these fascinating proteins further and to explore their potential use as surface modifying agents.

[1] Bayry J, Aimanianda V, Guijarro JI, Sunde M, Latgé J-P: Hydrophobins—Unique Fungal Proteins. PLoS Pathog, 8 (5):e1002700(2012)  doi:10.1371/journal.ppat.1002700
[2] Pham CLL, Rey A, Lo V, Soulès M, Ren Q, Meisl G, Knowles TPJ, Kwan AH, Sunde M: Self-assembly of MPG1, a hydrophobin protein from the rice blast fungus that forms functional amyloid coatings, occurs by a surface-driven mechanism. Scientific Reports, 6:25288(2016)  doi:10.1038/srep25288
[3] Wösten HAB, van Wetter M-A, Lugones LG, van der Mei HC, Busscher HJ, Wessels JGH: How a fungus escapes the water to grow into the air. Current Biology, 9 (2):85-88(1999)  doi:10.1016/S0960-9822(99)80019-0
[4] Lo VC, Ren Q, Pham CLL, Morris VK, Kwan AH, Sunde M: Fungal Hydrophobin Proteins Produce Self-Assembling Protein Films with Diverse Structure and Chemical Stability. Nanomaterials, 4 (3):827-843(2014)  doi:10.3390/nano4030827
[5] Macindoe I, Kwan AH, Ren Q, Morris VK, Yang W, Mackay JP, Sunde M: Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS. Proc Natl Acad Sci USA, 109 (14):E804-811(2012)  doi:10.1073/pnas.1114052109
[6] Morris VK, Ren Q, Macindoe I, Kwan AH, Byrne N, Sunde M: Recruitment of class I hydrophobins to the air:water interface initiates a multi-step process of functional amyloid formation. J Biol Chem:jbc.M110.214197(2011)  doi:10.1074/jbc.M110.214197
[7] Ren Q, Kwan AH, Sunde M: Solution structure and interface-driven self-assembly of NC2, a new member of the Class II hydrophobin proteins. Proteins, 82 (6):990-1003(2014)  doi:10.1002/prot.24473
[8] Stalder AF, Kulik G, Sage D, Barbieri L, Hoffmann P: A snake-based approach to accurate determination of both contact points and contact angles. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 286 (1):92-103(2006)  doi:10.1016/j.colsurfa.2006.03.008
[9] Vazquez G, Alvarez E, Navaza JM: Surface Tension of Alcohol Water + Water from 20 to 50 .degree.C. Journal of Chemical & Engineering Data, 40:611-614(1995)  doi:10.1021/je00019a016

Victor Lo1, Ann H. Kwan2, Margaret Sunde1

1School of Medicine, University of Sydney,  Australia
2School of Life and Environmental Sciences, University of Sydney,  Australia

Victor Lo

School of Medicine
University of Sydney
Sydney , Australia


University of Sydney


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