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Diffusion of Hydrophobin Proteins in Solution and Interactions with a Graphite Surface Paolo Mereghetti1,2* and Rebecca C Wade1*

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Diffusion of Hydrophobin Proteins in Solution and Interactions with a Graphite Surface Paolo Mereghetti1,2* and Rebecca C Wade1* Mereghetti and Wade BMC Biophysics 2011, 4:9 http://www.biomedcentral.com/2046-1682/4/9 RESEARCHARTICLE Open Access Diffusion of hydrophobin proteins in solution and interactions with a graphite surface Paolo Mereghetti1,2* and Rebecca C Wade1* Abstract Background: Hydrophobins are small proteins produced by filamentous fungi that have a variety of biological functions including coating of spores and surface adhesion. To accomplish these functions, they rely on unique interface-binding properties. Using atomic-detail implicit solvent rigid-body Brownian dynamics simulations, we studied the diffusion of HFBI, a class II hydrophobin from Trichoderma reesei, in aqueous solution in the presence and absence of a graphite surface. Results: In the simulations, HFBI exists in solution as a mixture of monomers in equilibrium with different types of oligomers. The oligomerization state depends on the conformation of HFBI. When a Highly Ordered Pyrolytic Graphite (HOPG) layer is present in the simulated system, HFBI tends to interact with the HOPG layer through a hydrophobic patch on the protein. Conclusions: From the simulations of HFBI solutions, we identify a tetrameric encounter complex stabilized by non-polar interactions between the aliphatic residues in the hydrophobic patch on HFBI. After the formation of the encounter complex, a local structural rearrangement at the protein interfaces is required to obtain the tetrameric arrangement seen in HFBI crystals. Simulations performed with the graphite surface show that, due to a combination of a geometric hindrance and the interaction of the aliphatic sidechains with the graphite layer, HFBI proteins tend to accumulate close to the hydrophobic surface. Background amino-acid sequence [1]. This classification is also Hydrophobins are small (7-15 kDa) proteins produced consistent with other properties. In particular, class I by filamentous fungi [1]. They perform a range of hydrophobins are more resistant to dissociation using sol- biological roles including coating of spores and surface vents and detergents than class II hydrophobins. Further- adhesion [2,3]. Except for Botrytis cinerea, where their more, class I hydrophobins tend to form rodlet-like function is unknown [4], hydrophobins lower the sur- aggregates at interfaces, whereas class II hydrophobins do face tension of water so that fungal hyphae can pene- not. Although, the different types of hydrophobins show a trate the air-water interface and grow outside aqueous great variability in aminoacid sequence (with sequence media [5]. To carry out these functions, they rely on identity sometimes as low as 30% [7]), they all present a unique surface/interface binding properties [1,3,6-8]. characteristic pattern of four disulfide bridges formed Besides their peculiar surface properties, which make by eight conserved cysteines [11]. This disulfide bridge them the most powerful surface-active proteins known pattern is common to all known class I and class II hydro- [3], they also display unusual behaviour in solution as phobins. For a recent review on hydrophobins, see ref. [3]. they form different kinds of oligomers, depending on The hydrophobin HFBI from Trichoderma reesei, the conditions and on the hydrophobin type [9,10]. which will be considered in this study, belongs to class Hydrophobins have been divided into two classes, class I II hydrophobins and its three-dimensional structure has and class II, based on the hydropathy profile of the been determined by X-ray crystallography by Hakanpää et al. at 2.1 Å resolution [12]. HFBI has an amphipathic ≈ 2 * Correspondence: [email protected]; [email protected] structure, with a large ( 750 Å ) solvent-exposed hydro- 1Heidelberg Institute for Theoretical Studies (HITS) gGmbH, Schloß- phobic patch containing aliphatic residues and a hydro- Wolfsbrunnenweg 35, 69118 Heidelberg, Germany philic region composed of polar residues. It is likely that Full list of author information is available at the end of the article © 2011 Mereghetti and Wad; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mereghetti and Wade BMC Biophysics 2011, 4:9 Page 2 of 11 http://www.biomedcentral.com/2046-1682/4/9 the unfavorable exposure of many hydrophobic residues described in the Methods section, is that the four chains to the aqueous solvent, is compensated by the four in the HBFI tetramer observed in the asymmetric unit disulfide bridges [3,9]. of the crystal structure can be divided into two groups In the crystal structure, HFBI forms homotetramers. based on the conformation of the second b-hairpin In solutions, HFBI forms oligomers in a concentration- (loop 60-66) and on their electrostatic potential [12]. dependent manner, and in particular, it has been shown Oligomerization that dimers and tetramers are present in aqueous Using the definition of oligomers described in the solution at protein concentrations (2-20 g/L) [13]. At Methods section, we computed the fractions of HFBI protein concentrations below 2 g/L, HFBI is monomeric molecules in different oligomeric states (from monomers in solution [13,14]. to pentamers) observed in the simulations (see Figure 1). Hydrophobins adsorb on to various types of surfaces, Considering the simulations performed with conforma- forming regular structures (e.g. hexagonal patterns) or tion A only (Figure 1A), it can be observed that, randomly aligned rodlets [3,8,10,15-17]. In a growing whereas the fraction of dimers is approximately con- number of works, hydrophobins are applied for surface stant, the fractions of higher order oligomers increases modification and in biosensor development [17-20]. The with the protein concentration. surface and interfacial activity of HFBI proteins has For the mixed chain A and chain B simulations, it can been studied from experimental [8,13,16] and theoretical be seen (Figure 1B) that the fractions of oligomers are [21,22] points of view. From these studies, a general generally lower than in the chain A only simulations. model for how hydrophobins function has emerged. Moreover, all the oligomer fractions (monomers to pen- Hydrophobins are soluble in aqueous solution and they tamers) show a concentration dependence. This suggests form different types of oligomers. Close to an interface, that oligomerization depends on a structural rearrange- the oligomers dissociate and adsorb on to the surface. ment of HBFI, as was previously proposed [12]. In parti- The secondary structure of HFBI does not change upon cular, Hakanpää et al. explained the different adsorption or self-assembly [8,23]. However minor conformations found in the crystal structure as resulting changes in the orientations of sidechains [8] or loops from a structural change after HBFI tetramer formation [12] do occur. It has been suggested that these local [12]. Analyzing the tetramers formed in the inhomoge- rearrangements and, in particular, the conformational neous system, we find that chain B tends to disfavour change of loop 60-66, are induced by multimer forma- the formation of tetramers. This is shown in Figure 2 tion [12]. where the fractions of the different types of tetramer The aim of this work is to shed light on the mechan- formed are shown. The two conformations essentially isms of self-association of HFBI in solution and its differ only in the structure of the second b-hairpin (loop adsorption onto a hydrophobic surface. A detailed 60-66), which in chain B is present in a solvent-exposed understanding of these mechanisms can be relevant not conformation, as well as in the orientation of some only for a better understanding of the biological sidechains. As was mention in ref. [12], it would not be function of hydrophobins but also for the potential bio- possible to form the tetramers observed in the crystal technological application of these macromolecules. structure if all the monomers were in the chain B In the next section, we discuss the results obtained conformation because of steric clashes of the extended from simulations of HFBI solutions at different concen- conformation of loop 60-66. Despite the structural rigid- trations. Then, results on the interaction of HFBI solu- ity of HFBI due to the four disulfide bridges, minor tions with a graphite surface are presented. structural changes occur, and seem to be important for the oligomerization process. It is not clear whether the Results and Discussion process of formation of the tetramers can be cast as an Solution properties of HFBI induced fit or a conformational selection model. This The properties of HFBI in aqueous solution were question could be addressed by performing all-atom studied by means of simulations at protein concentra- molecular dynamics simulations of the tetrameric tionsof2,5,10and20g/L.Accordingtotheexperi- encounter complexes generated in our Brownian mental conditions used in ref. [14], a pH of 5 and ionic dynamics simulations. strength of 50 mM were assumed. Two sets of simula- Tetrameric encounter complexes tions were done at each protein concentration. In one From the simulations, all tetramers were collected from set, all proteins were in conformation A (corresponding snapshots at 1 ns time intervals and then clustered to chain A in the crystal structure) while in the second to identify the structures of the tetramer that occur set, a mixture of 50% conformation A and 50% confor- with the highest probabilities in the simulations. In mation B (corresponding to chain B in the crystal Figure 3A, B, C, structures corresponding to the cen- structure) was simulated. The reason for this, as troids of the first three most populated clusters obtained Mereghetti and Wade BMC Biophysics 2011, 4:9 Page 3 of 11 http://www.biomedcentral.com/2046-1682/4/9 Figure 1 Oligomerization of HFBI in aqueous solutions. Fractions of monomeric c1 and higher oligomeric states cn observed in the simulations.
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