Published: March 17, 2011 r2011 American Chemical Society 4481 dx.doi.org/10.1021/la2001943 | Langmuir 2011, 27, 4481–4488 ARTICLE pubs.acs.org/Langmuir Self-Assembled Bilayers from the Protein HFBII Hydrophobin: Nature of the Adhesion Energy Elka S. Basheva, † Peter A. Kralchevsky,* ,† Krassimir D. Danov, † Simeon D. Stoyanov, ‡ Theo B. J. Blijdenstein, ‡ Eddie G. Pelan, ‡ and Alex Lips § † Department of Chemical Engineering, Faculty of Chemistry, Sofia University, 1164 Sofia, Bulgaria ‡ Unilever Research & Development, 3133AT Vlaardingen, The Netherlands § Unilever Research & Development, Port Sunlight, Wirral, Merseyside CH63 3JW, United Kingdom b S Supporting Information 1. INTRODUCTION The hydrophobins represent a class of relatively small proteins (about 100 amino acid residues) that are produced by filamen- tous fungi, including the well-known button mushrooms. In nature, these self-assembled films coat fungal structures and mediate their attachment to surfaces. Hydrophobins from the fungus Trichoderma reesei have been isolated, purified, and extensively studied, including the hydrophobin HFBII, 1,2 which is used in the present paper. The structure of HFBII determined from crystallized samples 3 shows that it is a single domain protein with dimensions of 2.4  2.7  3.0 nm 3 . In aqueous solutions, the class II hydrophobins form dimers (at low concentrations), whereas tetramers are the dominant assemblies at mg/mL concentrations. 47 It was shown that the oligomerization in- creases with the rise of protein concentration. 5 At the surface of water, HFBII forms self-assembled monolayers. 7,8 The adsorp- tion of this protein is not accompanied by changes in its secondary structure and ultrastructure. 7,9 Detailed reviews on the properties of hydrophobins have been published. 1012 Their special properties have found applications as stabilizers of foams and emulsions, 1316 as coating agents for surface modification, 1721 and for immobilization of functional molecules at various surfaces. 17,18,2224 In this Article we report results from the investigation of HFBII stabilized free foam films. We established that the drainage of such a film ends with the formation of a 6 nm thick film, which consists of two layers of protein molecules; that is, it is a self-assembled bilayer (S-bilayer). The analysis of the driving force of its appearance brings information about the origin of the adhesive action of HFBII. In section 2, we describe the experi- mental methods. In section 3, experimental results for the effects of pH and various electrolytes are presented. Finally, in section 4, based on the obtained results, we discuss the nature of the adhesion energy. Additional details are appended as Supporting Information, including data for the effect of cations and anions on the S-bilayer formation (Appendix A), theoretical derivation of the elliptic approximation for the meniscus profile (Appendix B), and theoretical analysis of the long-range branch of disjoining pressure isotherms (Appendix C). 2. METHODS 2.1. Hydrophobin Sample Production. We used a HFBII sample produced from yeast fermentation and purified according to procedures described in detail elsewhere. 25 Briefly, Saccharomyces cerevisiae strain CEN.PK338 (gal1:URA3, leu2, ura3, pmt1) carried a multicopy integration vector, integrated at the rDNA locus, containing the protein coding sequence of Trichoderma reesei HFBII, linked to the S. cerevisiae SUC2 signal sequence and under control of the GAL7 pro- moter and leu2d selectable marker to maintain a high copy number. 26 The strain was grown in fed batch fermentations. 27 The cells were removed by centrifugation and filtration over a 0.2 μm filter, and the supernatant containing the HFBII was freeze-dried. The pH of the Received: January 16, 2011 Revised: March 4, 2011 ABSTRACT: The hydrophobins are a class of amphiphilic proteins which spontaneously adsorb at the air/water interface and form elastic membranes of high mechanical strength as compared to other proteins. The mechanism of hydrophobin adhesion is of interest for fungal biology and for various applications in electronics, medicine, and food industry. We established that the drainage of free foam films formed from HFBII hydrophobin solutions ends with the appearance of a 6 nm thick film, which consists of two layers of protein molecules, that is, it is a self-assembled bilayer (S-bilayer), with hydrophilic domains pointing inward and hydrophobic domains pointing outward. Its formation is accompanied by a considerable energy gain, which is much greater than that typically observed with free liquid films. The experiments at different pH show that this attraction between the “hydrophilic” parts of the HFBII molecules is dominated by the short-range hydrophobic interaction rather than by the patch-charge electrostatic attraction.