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1.  Hydrophobin Film Structure for HFBI and HFBII and Mechanism for Accelerated Film Formation 
PLoS Computational Biology  2014;10(7):e1003745.
Hydrophobins represent an important group of proteins from both a biological and nanotechnological standpoint. They are the means through which filamentous fungi affect their environment to promote growth, and their properties at interfaces have resulted in numerous applications. In our study we have combined protein docking, molecular dynamics simulation, and electron cryo-microscopy to gain atomistic level insight into the surface structure of films composed of two class II hydrophobins: HFBI and HFBII produced by Trichoderma reesei. Together our results suggest a unit cell composed of six proteins; however, our computational results suggest P6 symmetry, while our experimental results show P3 symmetry with a unit cell size of 56 Å. Our computational results indicate the possibility of an alternate ordering with a three protein unit cell with P3 symmetry and a smaller unit cell size, and we have used a Monte Carlo simulation of a spin model representing the hydrophobin film to show how this alternate metastable structure may play a role in increasing the rate of surface coverage by hydrophobin films, possibly indicating a mechanism of more general significance to both biology and nanotechnology.
Author Summary
Filamentous fungi release a specific type of protein, belonging to a protein family known as “hydrophobins” into their environment to control interfaces in a fashion that promotes growth. Such protein coatings are the mechanism that allows for the mycelia to grow out of the water and into the air. When these hydrophobins form films at the air-water interface and on the surface of solid objects immersed in water, they impart properties to those surfaces that has led to their use in a wide range of industrial applications. Of particular interest is the properties they impart to air liquid interfaces, and as a mechanism to bring protective materials to coat nanoparticles in nanotechnology applications. A more detailed knowledge of the structure of these surfaces will allow for augmentation of their function that is possible through genetic engineering of the hydrophobins themselves. In this study we have combined computational and experimental methods to develop atomistic level insight into the structure of this surface for two important hydrophobins: HFBI and HFBII of Trichoderma reesei. In addition to insight into the surface structure, we have uncovered an intriguing possible new mechanism for film formation, which may explain some of the striking properties of hydrophobin films, and could be extended to a more general mechanism.
doi:10.1371/journal.pcbi.1003745
PMCID: PMC4117420  PMID: 25079355
2.  Expression of a Fungal Hydrophobin in the Saccharomyces cerevisiae Cell Wall: Effect on Cell Surface Properties and Immobilization 
The aim of this work was to modify the cell surface properties of Saccharomyces cerevisiae by expression of the HFBI hydrophobin of the filamentous fungus Trichoderma reesei on the yeast cell surface. The second aim was to study the immobilization capacity of the modified cells. Fusion to the Flo1p flocculin was used to target the HFBI moiety to the cell wall. Determination of cell surface characteristics with contact angle and zeta potential measurements indicated that HFBI-producing cells are more apolar and slightly less negatively charged than the parent cells. Adsorption of the yeast cells to different commercial supports was studied. A twofold increase in the binding affinity of the hydrophobin-producing yeast to hydrophobic silicone-based materials was observed, while no improvement in the interaction with hydrophilic carriers could be seen compared to that of the parent cells. Hydrophobic interactions between the yeast cells and the support are suggested to play a major role in attachment. Also, a slight increase in the initial adsorption rate of the hydrophobin yeast was observed. Furthermore, due to the engineered cell surface, hydrophobin-producing yeast cells were efficiently separated in an aqueous two-phase system by using a nonionic polyoxyethylene detergent, C12-18EO5.
doi:10.1128/AEM.68.7.3385-3391.2002
PMCID: PMC126783  PMID: 12089019
3.  Structure-Function Relationships in Hydrophobins: Probing the Role of Charged Side Chains 
Applied and Environmental Microbiology  2013;79(18):5533-5538.
Hydrophobins are small fungal proteins that are amphiphilic and have a strong tendency to assemble at interfaces. By taking advantage of this property, hydrophobins have been used for a number of applications: as affinity tags in protein purification, for protein immobilization, such as in foam stabilizers, and as dispersion agents for insoluble drug molecules. Here, we used site-directed mutagenesis to gain an understanding of the molecular basis of their properties. We especially focused on the role of charged amino acids in the structure of hydrophobins. For this purpose, fusion proteins consisting of Trichoderma reesei hydrophobin I (HFBI) and the green fluorescent protein (GFP) that contained various combinations of substitutions of charged amino acids (D30, K32, D40, D43, R45, K50) in the HFBI structure were produced. The effects of the introduced mutations on binding, oligomerization, and partitioning were characterized in an aqueous two-phase system. It was found that some substitutions caused better surface binding and reduced oligomerization, while some showed the opposite effects. However, all mutations decreased partitioning in surfactant systems, indicating that the different functions are not directly correlated and that partitioning is dependent on finely tuned properties of hydrophobins. This work shows that not all functions in self-assembly are connected in a predictable way and that a simple surfactant model for hydrophobin function is insufficient.
doi:10.1128/AEM.01493-13
PMCID: PMC3754166  PMID: 23835172
4.  Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins 
BMC Biotechnology  2013;13:40.
Background
Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum.
Results
The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants.
Conclusion
The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags increase the accumulation levels of the recombinant protein and induce the formation of PBs regardless of the cultivar used. However, a specific level of recombinant protein accumulation needs to be reached for PBs to form.
doi:10.1186/1472-6750-13-40
PMCID: PMC3659085  PMID: 23663656
Protein body; Protein body formation; Elastin-like polypeptide; ELP; Hydrophobin I; HFBI; Tobacco; Transgenic expression; Molecular farming; Green fluorescent protein
5.  The Contribution of Polystyrene Nanospheres towards the Crystallization of Proteins 
PLoS ONE  2009;4(1):e4198.
Background
Protein crystallization is a slow process of trial and error and limits the amount of solved protein structures. Search of a universal heterogeneous nucleant is an effort to facilitate crystallizability of proteins.
Methodology
The effect of polystyrene nanospheres on protein crystallization were tested with three commercial proteins: lysozyme, xylanase, xylose isomerase, and with five research target proteins: hydrophobins HFBI and HFBII, laccase, sarcosine dimethylglycine N-methyltransferase (SDMT), and anti-testosterone Fab fragment 5F2. The use of nanospheres both in screening and as an additive for known crystallization conditions was studied. In screening, the addition of an aqueous solution of nanosphere to the crystallization drop had a significant positive effect on crystallization success in comparison to the control screen. As an additive in hydrophobin crystallization, the nanospheres altered the crystal packing, most likely due to the amphiphilic nature of hydrophobins. In the case of laccase, nanospheres could be used as an alternative for streak-seeding, which insofar had remained the only technique to produce high-diffracting crystals. With methyltransferase SDMT the nanospheres, used also as an additive, produced fewer, larger crystals in less time. Nanospheres, combined with the streak-seeding method, produced single 5F2 Fab crystals in shorter equilibration times.
Conclusions
All in all, the use of nanospheres in protein crystallization proved to be beneficial, both when screening new crystallization conditions to promote nucleation and when used as an additive to produce better quality crystals, faster. The polystyrene nanospheres are easy to use, commercially available and close to being inert, as even with amphiphilic proteins only the crystal packing is altered and the nanospheres do not interfere with the structure and function of the protein.
doi:10.1371/journal.pone.0004198
PMCID: PMC2615210  PMID: 19145241
6.  Modification of Hydrophilic and Hydrophobic Surfaces Using an Ionic-Complementary Peptide 
PLoS ONE  2007;2(12):e1325.
Ionic-complementary peptides are novel nano-biomaterials with a variety of biomedical applications including potential biosurface engineering. This study presents evidence that a model ionic-complementary peptide EAK16-II is capable of assembling/coating on hydrophilic mica as well as hydrophobic highly ordered pyrolytic graphite (HOPG) surfaces with different nano-patterns. EAK16-II forms randomly oriented nanofibers or nanofiber networks on mica, while ordered nanofibers parallel or oriented 60° or 120° to each other on HOPG, reflecting the crystallographic symmetry of graphite (0001). The density of coated nanofibers on both surfaces can be controlled by adjusting the peptide concentration and the contact time of the peptide solution with the surface. The coated EAK16-II nanofibers alter the wettability of the two surfaces differently: the water contact angle of bare mica surface is measured to be <10°, while it increases to 20.3±2.9° upon 2 h modification of the surface using a 29 µM EAK16-II solution. In contrast, the water contact angle decreases significantly from 71.2±11.1° to 39.4±4.3° after the HOPG surface is coated with a 29 µM peptide solution for 2 h. The stability of the EAK16-II nanofibers on both surfaces is further evaluated by immersing the surface into acidic and basic solutions and analyzing the changes in the nanofiber surface coverage. The EAK16-II nanofibers on mica remain stable in acidic solution but not in alkaline solution, while they are stable on the HOPG surface regardless of the solution pH. This work demonstrates the possibility of using self-assembling peptides for surface modification applications.
doi:10.1371/journal.pone.0001325
PMCID: PMC2117347  PMID: 18091996
7.  Purifying selection and birth-and-death evolution in the class II hydrophobin gene families of the ascomycete Trichoderma/Hypocrea 
Background
Hydrophobins are proteins containing eight conserved cysteine residues that occur uniquely in mycelial fungi. Their main function is to confer hydrophobicity to fungal surfaces in contact with air or during attachment of hyphae to hydrophobic surfaces of hosts, symbiotic partners or themselves resulting in morphogenetic signals. Based on their hydropathy patterns and solubility characteristics, hydrophobins are divided into two classes (I and II), the latter being found only in ascomycetes.
Results
We have investigated the mechanisms driving the evolution of the class II hydrophobins in nine species of the mycoparasitic ascomycetous genus Trichoderma/Hypocrea, using three draft sequenced genomes (H. jecorina = T. reesei, H. atroviridis = T. atroviride; H. virens = T. virens) an additional 14,000 ESTs from six other Trichoderma spp. (T. asperellum, H. lixii = T. harzianum, T. aggressivum var. europeae, T. longibrachiatum, T. cf. viride). The former three contained six, ten and nine members, respectively. Ten is the highest number found in any ascomycete so far. All the hydrophobins we examined had the conserved four beta-strands/one helix structure, which is stabilized by four disulfide bonds. In addition, a small number of these hydrophobins (HFBs)contained an extended N-terminus rich in either proline and aspartate, or glycine-asparagine. Phylogenetic analysis reveals a mosaic of terminal clades containing duplicated genes and shows only three reasonably supported clades. Calculation of the ratio of differences in synonymous vs. non-synonymous nucleotide substitutions provides evidence for strong purifying selection (KS/Ka >> 1). A genome database search for class II HFBs from other ascomycetes retrieved a much smaller number of hydrophobins (2–4) from each species, and most were from Sordariomycetes. A combined phylogeny of these sequences with those of Trichoderma showed that the Trichoderma HFBs mostly formed their own clades, whereas those of other Sordariomycetes occurred in shared clades.
Conclusion
Our study shows that the genus Trichoderma/Hypocrea has a proliferated arsenal of class II hydrophobins which arose by birth-and-death evolution followed by purifying selection.
doi:10.1186/1471-2148-8-4
PMCID: PMC2253510  PMID: 18186925
8.  Lack of evidence for a role of hydrophobins in conferring surface hydrophobicity to conidia and hyphae of Botrytis cinerea 
BMC Microbiology  2011;11:10.
Background
Hydrophobins are small, cysteine rich, surface active proteins secreted by filamentous fungi, forming hydrophobic layers on the walls of aerial mycelia and spores. Hydrophobin mutants in a variety of fungi have been described to show 'easily wettable' phenotypes, indicating that hydrophobins play a general role in conferring surface hydrophobicity to aerial hyphae and spores.
Results
In the genome of the grey mould fungus Botrytis cinerea, genes encoding three hydrophobins and six hydrophobin-like proteins were identified. Expression analyses revealed low or no expression of these genes in conidia, while some of them showed increased or specific expression in other stages, such as sclerotia or fruiting bodies. Bhp1 belongs to the class I hydrophobins, whereas Bhp2 and Bhp3 are members of hydrophobin class II. Single, double and triple hydrophobin knock-out mutants were constructed by consecutively deleting bhp1, bhp2 and bhp3. In addition, a mutant in the hydrophobin-like gene bhl1 was generated. The mutants were tested for germination and growth under different conditions, formation of sclerotia, ability to penetrate and infect host tissue, and for spore and mycelium surface properties. Surprisingly, none of the B. cinerea hydrophobin mutants showed obvious phenotypic defects in any of these characters. Scanning electron microscopy of the hydrophobic conidial surfaces did not reveal evidence for the presence of typical hydrophobin 'rodlet' layers.
Conclusions
These data provide evidence that in B. cinerea, hydrophobins are not involved in conferring surface hydrophobicity to conidia and aerial hyphae, and challenge their universal role in filamentous fungi. The function of some of these proteins in sclerotia and fruiting bodies remains to be investigated.
doi:10.1186/1471-2180-11-10
PMCID: PMC3032640  PMID: 21232149
9.  Controlling the dispersion of supported polyoxometalate heterogeneous catalysts: impact of hybridization and the role of hydrophilicity–hydrophobicity balance and supramolecularity 
Summary
The hybridization of polyoxometalates (POMs) through an organic–inorganic association offers several processing advantages in the design of heterogeneous catalysts. A clear understanding of the organization of these hybrid materials on solid surfaces is necessary to optimise their properties. Herein, we report for the first time the organization of Keggin phosphotungstic [PW12O40]3− and Wells–Dawson (WD) phosphomolybdic [P2Mo18O62]6− anions deposited on mica (hydrophilic), and highly oriented pyrolytic graphite (HOPG) (hydrophobic) surfaces. Next, the supramolecular organization of the organic–inorganic hybrid materials formed from the association of POM anions and dimethyldioctadecylammonium bromide (DODA) is investigated as a function of the hydrophilic or hydrophobic nature of the surfaces. The height of the Keggin-POM anions, measured with tapping mode (TM-AFM) is always in good agreement with the molecular dimension of symmetric Keggin-POM anions (ca. 1 nm). However, the asymmetric WD-POM anions form monolayer assemblies on the surfaces with the orientation of their long molecular axis (ca. 1.6 nm) depending on the hydrophilic or hydrophobic properties of the substrate. Namely, the long axis is parallel on mica, and perpendicular on HOPG. When hybridized with DODA, the organization of the hybrid material is dictated by the interaction of the alkyl side chains of DODA with the substrate surface. On HOPG, the DODA–POM hybrid forms small domains of epitaxially arranged straight nanorod structures with their orientation parallel to each other. Conversely, randomly distributed nanospheres are formed when the hybrid material is deposited on freshly cleaved mica. Finally, a UV–ozone treatment of the hybrid material allows one to obtain highly dispersed isolated POM entities on both hydrophilic and hydrophobic surfaces. The hybridization strategy to prevent the clustering of POMs on various supports would enable to develop highly dispersed POM-based heterogeneous catalysts with enhanced functionalities.
doi:10.3762/bjnano.5.185
PMCID: PMC4222433  PMID: 25383286
atomic force microscopy; heterogeneous hybrid catalyst; organic–inorganic hybrid materials; polyoxometalates; supramolecular organization
10.  Cerato-platanins: a fungal protein family with intriguing properties and application potential 
Applied Microbiology and Biotechnology  2014;98(11):4795-4803.
Cerato-platanin proteins are small, secreted proteins with four conserved cysteines that are abundantly produced by filamentous fungi with all types of lifestyles. These proteins appear to be readily recognized by other organisms and are therefore important factors in interactions of fungi with other organisms, e.g. by stimulating the induction of defence responses in plants. However, it is not known yet whether the main function of cerato-platanin proteins is associated with these fungal interactions or rather a role in fungal growth and development. Cerato-platanin proteins seem to unify several biochemical properties that are not found in this combination in other proteins. On one hand, cerato-platanins are carbohydrate-binding proteins and are able to bind to chitin and N-acetylglucosamine oligosaccharides; on the other hand, they are able to self-assemble at hydrophobic/hydrophilic interfaces and form protein layers, e.g. on the surface of aqueous solutions, thereby altering the polarity of solutions and surfaces. The latter property is reminiscent of hydrophobins, which are also small, secreted fungal proteins, but interestingly, the surface-activity-altering properties of cerato-platanins are the opposite of what can be observed for hydrophobins. The so far known biochemical properties of cerato-platanin proteins are summarized in this review, and potential biotechnological applications as well as implications of these properties for the biological functions of cerato-platanin proteins are discussed.
doi:10.1007/s00253-014-5690-y
PMCID: PMC4024134  PMID: 24687753
Cerato-platanin; Chitin; Self-assembly; Protein layer; Hydrophobin; Expansin
11.  Interface Properties of Organic para-Hexaphenyl/α-Sexithiophene Heterostructures Deposited on Highly Oriented Pyrolytic Graphite 
Langmuir  2013;29(47):14444-14450.
It was recently reported, that heterostructures of para-hexaphenyl (p-6P) and α-sexithiophene (6T) deposited on muscovite mica exhibit the intriguing possibility to prepare lasing nanofibers of tunable emission wavelength. For p-6P/6T heterostructures, two different types of 6T emission have been observed, namely, the well-known red emission of bulk 6T crystals and additionally a green emission connected to the interface between p-6P and 6T. In this study, the origin of the green fluorescence is investigated by photoelectron spectroscopy (PES). As a prerequisite, it is necessary to prepare structurally similar organic crystals on a conductive surface, which leads to the choice of highly oriented pyrolytic graphite (HOPG) as a substrate. The similarity between p-6P/6T heterostructures on muscovite mica and on HOPG is evidenced by X-ray diffraction (XRD), scanning force microscopy (SFM), and optical spectroscopy. PES measurements show that the interface between p-6P and 6T crystals is sharp on a molecular level without any sign of interface dipole formation or chemical interaction between the molecules. We therefore conclude that the different emission colors of the two 6T phases are caused by different types of molecular aggregation.
doi:10.1021/la402242b
PMCID: PMC3842851  PMID: 24156627
12.  Identification of new members of hydrophobin family using primary structure analysis 
BMC Bioinformatics  2006;7(Suppl 4):S16.
Background
Hydrophobins are fungal proteins that can turn into amphipathic membranes at hydrophilic/hydrophobic interfaces by self-assembly. The assemblages by Class I hydrophobins are extremely stable and possess the remarkable ability to change the polarity of the surface. One of its most important industrial applications is its usage as paint. Without detailed knowledge of the 3D structure and self-assembly principles of hydrophobins, it is difficult to make significant progress in furthering its research.
Results
In order to provide useful information to hydrophobin researchers, we analyzed primary structure of hydrophobins to gain more insight about these proteins. In this paper, we presented an in-depth primary sequence analysis using batch BLAST search of the database, sequence filtering by programming and motif finding by MEME. We used batch BLAST to find similar sequences in the NCBI nr database. Then we used MEME to find out motifs. Based on the newly found motifs and the well-known C-CC-C-C-CC-C pattern we used MAST to search the entire nr database. At the end, domain search and phylogenetic analysis were conducted to confirm the result. After searching the nr database with the new PSSM-format motifs identified by MEME, many sequences from various species were found by MAST. Filtering process by pattern, domain and length left 9 qualified candidates.
Conclusion
All of 9 newly identified potential hydrophobins possess the common pattern and hydrophobin domain. From the multiple sequence alignment result, we can see that some of them are grouped very close to other known hydrophobins, which means their phylogenetic relationship is very close and it is highly plausible that they are indeed hydrophobin proteins.
doi:10.1186/1471-2105-7-S4-S16
PMCID: PMC1780129  PMID: 17217508
13.  Production and characterization of in planta transiently produced polygalacturanase from Aspergillus niger and its fusions with hydrophobin or ELP tags 
BMC Biotechnology  2014;14:59.
Background
Pectinases play an important role in plant cell wall deconstruction and have potential in diverse industries such as food, wine, animal feed, textile, paper, fuel, and others. The demand for such enzymes is increasing exponentially, as are the efforts to improve their production and to implement their use in several industrial processes. The goal of this study was to examine the potential of producing polygalacturonase I from Aspergillus niger in plants and to investigate the effects of subcellular compartmentalization and protein fusions on its accumulation and activity.
Results
Polygalacturonase I from Aspergillus niger (AnPGI) was transiently produced in Nicotiana benthamiana by targeting it to five different cellular compartments: apoplast, endoplasmic reticulum (ER), vacuole, chloroplast and cytosol. Accumulation levels of 2.5%, 3.0%, and 1.9% of total soluble protein (TSP) were observed in the apoplast, ER, and vacuole, respectively, and specific activity was significantly higher in vacuole-targeted AnPGI compared to the same enzyme targeted to the ER or apoplast. No accumulation was found for AnPGI when targeted to the chloroplast or cytosol. Analysis of AnPGI fused with elastin-like polypeptide (ELP) revealed a significant increase in the protein accumulation level, especially when targeted to the vacuole where the protein doubles its accumulation to 3.6% of TSP, while the hydrophobin (HFBI) fusion impaired AnPGI accumulation and both tags impaired activity, albeit to different extents. The recombinant protein showed activity against polygalacturonic acid with optimum conditions at pH 5.0 and temperature from 30 to 50°C, depending on its fusion. In vivo analysis of reducing sugar content revealed a higher release of reducing sugars in plant tissue expressing recombinant AnPGI compared to wild type N. benthamiana leaves.
Conclusion
Our results demonstrate that subcellular compartmentalization of enzymes has an impact on both the target protein accumulation and its activity, especially in the case of proteins that undergo post-translational modifications, and should be taken into consideration when protein production strategies are designed. Using plants to produce heterologous enzymes for the degradation of a key component of the plant cell wall could reduce the cost of biomass pretreatment for the production of cellulosic biofuels.
doi:10.1186/1472-6750-14-59
PMCID: PMC4083859  PMID: 24970673
Polygalacturonase; Aspergillus niger; Nicotiana benthamiana; Elastin-like polypeptide; Hydrophobin I; Cellulosic biofuels
14.  Molecular-scale Hydrophilicity Induced by Solute: Molecular-thick Charged Pancakes of Aqueous Salt Solution on Hydrophobic Carbon-based Surfaces 
Scientific Reports  2014;4:6793.
We directly observed molecular-thick aqueous salt-solution pancakes on a hydrophobic graphite surface under ambient conditions employing atomic force microscopy. This observation indicates the unexpected molecular-scale hydrophilicity of the salt solution on graphite surfaces, which is different from the macroscopic wetting property of a droplet standing on the graphite surface. Interestingly, the pancakes spontaneously displayed strong positively charged behavior. Theoretical studies showed that the formation of such positively charged pancakes is attributed to cation–π interactions between Na+ ions in the aqueous solution and aromatic rings on the graphite surface, promoting the adsorption of water molecules together with cations onto the graphite surface; i.e., Na+ ions as a medium adsorbed to the graphite surface through cation–π interactions on one side while at the same time bonding to water molecules through hydration interaction on the other side at a molecular scale. These findings suggest that actual interactions regarding carbon-based graphitic surfaces including those of graphene, carbon nanotubes, and biochar may be significantly different from existing theory and they provide new insight into the control of surface wettability, interactions and related physical, chemical and biological processes.
doi:10.1038/srep06793
PMCID: PMC4210940  PMID: 25348642
15.  Hydrophobin Vmh2–glucose complexes self-assemble in nanometric biofilms 
Hydrophobins are small proteins secreted by fungi, which self-assemble into amphipathic membranes at air–liquid or liquid–solid interfaces. The physical and chemical properties of some hydrophobins, both in solution and as a biofilm, are affected by poly or oligosaccharides. We have studied the interaction between glucose and the hydrophobin Vmh2 from Pleurotus ostreatus by spectroscopic ellipsometry (SE), atomic force microscopy (AFM) and water contact angle (WCA). We have found that Vmh2–glucose complexes forms a chemically stable biofilm, obtained by drop deposition on silicon, 1.6 nm thick and containing 35 per cent of glucose, quantified by SE. AFM highlighted the presence of nanometric rodlet-like aggregates (average height, width and length being equal to 3.6, 23.8 and 40 nm, respectively) on the biofilm surface, slightly different from those obtained in the absence of glucose (4.11, 23.9 and 64 nm). The wettability of a silicon surface, covered by the organic layer of Vmh2–glucose, strongly changed: WCA decreased from 90° down to 17°.
doi:10.1098/rsif.2012.0217
PMCID: PMC3427517  PMID: 22572031
surface modification; protein biofilm; spectroscopic ellipsometry; atomic force microscopy
16.  A Molecular Dynamics Study of the Behavior of Selected Nanoscale Building Blocks in a Gel-Phase Lipid Bilayer 
The journal of physical chemistry. B  2010;114(28):9165-9172.
The cellular membrane functions as a regulating barrier between the intracellular and extracellular regions. For a molecule to reach the interior of the cell from the extracellular fluid, it must diffuse across the membrane, via either active or passive transport. The rigid structure of lipid bilayers, which are a key component of cellular membranes, prohibit simple diffusion of most particles, while vital nutrients are transported to the interior by specific mechanisms, such as ion channels and transport proteins. Although the cellular membrane provides the cell with protection against unwanted toxins that may be in the extracellular medium, some foreign particles can reach the interior of the cell, resulting in irregularities in cellular function. This behavior is particularly noted for permeants with compact molecular structure, suggesting that common nanoscale building blocks, such as fullerenes, may enter into the interior of a cell. To gauge the propensity for such particles to cross the membrane, we have computed the Gibbs free energy of transfer along the axis normal to the bilayer surface for two nanoscale building blocks, C60 and a hydrogen-terminated polyhedral oligomeric silsequioxane (H-POSS) monomer, in a hydrated dipalmitoylphosphatidylcholine (DPPC) bilayer using molecular dynamics simulations and potential of mean force calculations. The studies show that C60 has a substantial energetic preference for the soft polymer region of the lipid bilayer system, below the water/bilayer interface, with a transition energy from bulk water of −19.8 kcal/mol. The transition of C60 from the bulk water to the center of the bilayer, while also energetically favorable, has to overcome a +5.9 kcal/mol energetic barrier in the hydrophobic lipid tail region. The H-POSS simulations indicate an energy minimum at the water-bilayer interface, with an energy of −10.9 kcal/mol; however, a local minimum of −2.7 kcal/mol is also observed in the hydrophobic dense-aliphatic region. The energy barrier seen in the hydrophobic core region of the C60 study is likely due to the significant penalty associated with inserting the relatively large particle into such a dense region. In contrast, while H-POSS is found to be subject to an energetic penalty upon insertion into the bilayer, the relatively small size of the H-POSS solute renders this penalty less significant. The energy barrier seen in the soft polymer region for the H-POSS monomer is primarily attributed to the lack of favorable solute-bilayer electrostatic interactions, which are present in the interfacial region, and fewer van der Waals interactions in the soft polymer region than the dense aliphatic region. These results indicate that C60 may partition into the organic phase of the DPPC/water system, given the favorable free energies in the soft polymer and dense aliphatic regions of the bilayer, while H-POSS is likely to partition near the water-bilayer interface, where the particle has low-energy electrostatic interactions with the polar head groups of the bilayer.
doi:10.1021/jp1039942
PMCID: PMC2925108  PMID: 20583770
DPPC; fullerene C60; POSS; nanoparticle; molecular dynamics; potential of mean force; Gibbs free energy; solubility
17.  Surface Modifications Created by Using Engineered Hydrophobins 
Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25 N-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.
doi:10.1128/AEM.68.3.1367-1373.2002
PMCID: PMC123772  PMID: 11872489
18.  Surface Confined Metallosupramolecular Architectures: Formation and STM Characterization 
Accounts of chemical research  2009;42(2):249-259.
CONSPECTUS
Metallosupramolecular compounds have attracted a great deal of attention over the past two decades largely because of their unique, highly-complex structural characteristics, the fact that they can be prepared with relative ease using coordination-driven self-assembly techniques, and their potential electronic, magnetic, optical, and catalytic properties. In particular, the use of electron-poor square planar Pt(II) transition metals in conjunction with rigid, electron-rich pyridyl donors had enabled the spontaneous self-assembly of a rich library of 2D metallacyclic and 3D metallacage assemblies via the directional-bonding approach. With the tremendous progress that has been made in the preparation and characterization of metallosupramolecules, much attention is now being turned toward fully exploring and developing their materials properties.
Assembling metallosupramolecular compounds on solid supports represents a vitally important step toward developing their materials properties. Surfaces provide a means of uniformly aligning and orienting these highly symmetric metallacycles and metallacages, which increases the level of coherence between molecules above that which can be achieved in the solution phase and affords a means of integrating adlayers into a solid-state materials setting. The dynamic nature of kinetically labile Pt(II)-N coordination bonds, however, requires that deposition and imaging conditions be appropriate to retain the assemblies' stability. Toward these aims it is imperative to understand the factors that govern surface self-assembly and the interactions that influence the structure and stability of the resulting adlayer. Such insight can be obtained from Scanning Tunneling Microscopy (STM), which has emerged as a powerful technique for the imaging and characterization of self-assembled adlayers.
This account describes the means by which 2D rectangular and square metallacycles and 3D trigonal bipyrimidal and chiral trigonal prism metallacages can be deposited on Highly Oriented Pyrolytic Graphite (HOPG) and Au(111) substrates such that the assemblies remain intact and result in ordered adlayers. The effects of varying the size, symmetry, and dimensionality of supramolecular adsorbates, the choice of substrate, the use of a molecular template, and the effects of chirality have been investigated. These systematic investigations provide much insight into the various adsorbate-adsorbate and substrate-adsorbate interactions that largely determine the architecture of each assembly and affect their performance in a materials setting. Exhibiting the ability to rationally control adlayer formation and structure will greatly enhance the potential of these supramolecules to be used in a variety of applications such as in host-guest sensing/diagnostic systems, molecular electronic devices, and in heterogeneous stereoselective synthesis and catalysis.
doi:10.1021/ar800117j
PMCID: PMC2654282  PMID: 19072706
19.  Hydrophobin can prevent secondary protein adsorption on hydrophobic substrates without exchange 
Analytical and Bioanalytical Chemistry  2011;400(7):2031-2040.
By combining several surface analytical tools, we show that an adsorbed layer of the protein H*Protein B prevents the adsorption of secondary proteins bovine serum albumin, casein, or collagen at low-salinity conditions and at pH 8. H*Protein B is an industrially producible fusion protein of the hydrophobin family, known for its high interfacial activity. While applications of hydrophobin have been reported to facilitate adhesion of proteins under different pH conditions, careful analysis by quartz-crystal microbalance and ellipsometry prove that no additional adsorption can be found on top of the H*Protein B layer in this study. Surface analysis by X-ray photoelectron spectroscopy and secondary ion mass spectrometry proves that the hydrophobin layer stays intact even after hours of exposure to solutions of the secondary proteins and that no exchange of proteins can be detected.
Fig. 1The combination of surface analytical methods shows that an adsorbed layer of H*Protein B, a technically available representative of the hydrophobin family, prevents adsorption of secondary proteins at low-salinity conditions and pH 8. Due to the multi-method approach it can be proven that no exchange of the adsorbed hydrophobin protein is taking place
doi:10.1007/s00216-011-4902-x
PMCID: PMC3098362  PMID: 21461987
Hydrophobin; Protein adsorption; Secondary ion mass spectrometry; Quartz-crystal microbalance; X-ray photoelectron spectroscopy
20.  Molecular Engineering of Supramolecular Scaffold Coatings that Can Reduce Static Platelet Adhesion 
Novel supramolecular coatings that make use of low molecular weight ditopic monomers with guanine end groups are studied using fluid tapping AFM. These molecules assemble on highly oriented pyrolytic graphite (HOPG) from aqueous solutions to form nano-sized banding structures whose sizes can be systematically tuned at the nano-scale by tailoring the molecular structure of the monomers. The nature of the self-assembly in these systems has been studied through a combination of the self-assembly of structural derivatives and molecular modeling. Furthermore, we introduce the concept of using these molecular assemblies as scaffolds to organize functional groups on the surface. As a first demonstrationof this concept, scaffold monomers that contain a monomethyl triethyleneglycol branch were used to organize these “functional” units on a HOPG surface. These supramolecular grafted assemblies have been shown to be stable in biologically-relevant environments and even have the ability to significantly reduce static platelet adhesion.
doi:10.1021/ja0775927
PMCID: PMC2536642  PMID: 18177047
nanopattern; nanotechnology; surface assembly; supramolecular polymerization; surface thrombosis; biomaterial coating
21.  Preparation of monolayers of [MnIII6CrIII]3+ single-molecule magnets on HOPG, mica and silicon surfaces and characterization by means of non-contact AFM 
Nanoscale Research Letters  2011;6(1):486.
We report on the characterization of various salts of [MnIII6CrIII]3+ complexes prepared on substrates such as highly oriented pyrolytic graphite (HOPG), mica, SiO2, and Si3N4. [MnIII6CrIII]3+ is a single-molecule magnet, i.e., a superparamagnetic molecule, with a blocking temperature around 2 K. The three positive charges of [MnIII6CrIII]3+ were electrically neutralized by use of various anions such as tetraphenylborate (BPh4-), lactate (C3H5O3-), or perchlorate (ClO4-). The molecule was prepared on the substrates out of solution using the droplet technique. The main subject of investigation was how the anions and substrates influence the emerging surface topology during and after the preparation. Regarding HOPG and SiO2, flat island-like and hemispheric-shaped structures were created. We observed a strong correlation between the electronic properties of the substrate and the analyzed structures, especially in the case of mica where we observed a gradient in the analyzed structures across the surface.
doi:10.1186/1556-276X-6-486
PMCID: PMC3212000  PMID: 21824398
22.  Binding Preferences, Surface Attachment, Diffusivity, and Orientation of a Family 1 Carbohydrate-binding Module on Cellulose* 
The Journal of Biological Chemistry  2012;287(24):20603-20612.
Background: Family 1 carbohydrate-binding modules (CBMs) bind selectively to the hydrophobic surfaces of cellulose.
Results: Simulations have shown that the planar face of the CBM binds preferentially to the hydrophobic face.
Conclusion: Thermodynamic driving forces enable transfer of the CBM from the hydrophilic to hydrophobic surfaces.
Significance: Selectivity of CBM provides access of cellulases to active surfaces of cellulose.
Cellulase enzymes often contain carbohydrate-binding modules (CBMs) for binding to cellulose. The mechanisms by which CBMs recognize specific surfaces of cellulose and aid in deconstruction are essential to understand cellulase action. The Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase, Cel7A, is known to selectively bind to hydrophobic surfaces of native cellulose. It is most commonly suggested that three aromatic residues identify the planar binding face of this CBM, but several recent studies have challenged this hypothesis. Here, we use molecular simulation to study the CBM binding orientation and affinity on hydrophilic and hydrophobic cellulose surfaces. Roughly 43 μs of molecular dynamics simulations were conducted, which enables statistically significant observations. We quantify the fractions of the CBMs that detach from crystal surfaces or diffuse to other surfaces, the diffusivity along the hydrophobic surface, and the overall orientation of the CBM on both hydrophobic and hydrophilic faces. The simulations demonstrate that there is a thermodynamic driving force for the Cel7A CBM to bind preferentially to the hydrophobic surface of cellulose relative to hydrophilic surfaces. In addition, the simulations demonstrate that the CBM can diffuse from hydrophilic surfaces to the hydrophobic surface, whereas the reverse transition is not observed. Lastly, our simulations suggest that the flat faces of Family 1 CBMs are the preferred binding surfaces. These results enhance our understanding of how Family 1 CBMs interact with and recognize specific cellulose surfaces and provide insights into the initial events of cellulase adsorption and diffusion on cellulose.
doi:10.1074/jbc.M112.358184
PMCID: PMC3370244  PMID: 22496371
Cellulase; Molecular Dynamics; Molecular Modeling; Protein Dynamics; Protein Structure; Cel7A; Carbohydrate-binding Module; Cellulose; Hypocrea jecorina; Trichoderma reesei
23.  Identification and characterization of secreted and pathogenesis-related proteins in Ustilago maydis 
Molecular Genetics and Genomics   2007;279(1):27-39.
Interactions between plants and fungal pathogens require a complex interplay at the plant–fungus interface. Extracellular effector proteins are thought to play a crucial role in establishing a successful infection. To identify pathogenesis-related proteins in Ustilago maydis we combined the isolation of secreted proteins using a signal sequence trap approach with bioinformatic analyses and the subsequent characterization of knock-out mutants. We identified 29 secreted proteins including hydrophobins and proteins with a repetitive structure similar to the repellent protein Rep1. Hum3, a protein containing both, a hydrophobin domain and a repetitive Rep1-like region, is shown to be processed during passage through the secretory pathway. While single knock-outs of hydrophobin or repellent-like genes did not affect pathogenicity, we found a strong effect of a double knock-out of hum3 and the repetitive rsp1. Yeast-like growth, mating, aerial hyphae formation and surface hydrophobicity were unaffected in this double mutant. However, pathogenic development in planta stops early after penetration leading to a complete loss of pathogenicity. This indicates that Hum3 and Rsp1 are pathogenicity proteins that share an essential function in early stages of the infection. Our results demonstrate that focusing on secreted proteins is a promising way to discover novel pathogenicity proteins that might be broadly applied to a variety of fungal pathogens.
doi:10.1007/s00438-007-0291-4
PMCID: PMC2757621  PMID: 17917743
Hydrophobin; Repellent proteins; Phytopathogenic; Virulence factors; Signal sequence trap
24.  A Fluid Salt-Bridging Cluster and the Stabilization of p53 ⋆ 
Journal of molecular biology  2007;373(5):1334-1347.
p53 is a homotetrameric tumor suppressor protein that is found to be mutated in most human cancers. Some of these mutations, particularly mutations to R337 fall in the tetramerization domain and cause defects in tetramer formation leading to loss of function. Mutation to His at this site has been found to destabilize the tetramer in a pH-dependent fashion. In structures of the tet domain determined by crystallography, R337 from one monomer makes a salt bridge with D352 from another monomer, apparently helping to stabilize the tetramer. Here we present molecular dynamics simulations of wild-type p53 and the R337His mutant at several different pH and salt conditions. We find that the 337–352 salt bridge is joined by two other charged sidechains, R333 and E349. These four residues do not settle into a fixed pattern of salt-bridging, but continue to exchange salt-bridging partners on the nanosecond timescale throughout the simulation. This unusual system of fluid salt-bridging may explain the previous finding from alanine scanning experiments that R333 contributes significantly to protein stability even though, in the crystal structure it is extended outward into solvent. This extended conformation of R333 appears to be the result of a specific crystal contact and, this contact being absent in the simulation, R333 turns inward to join its interaction partners. When R337 is mutated to His but remains positively charged, it maintains the original interaction with D352, but the newly observed interaction with E349 is weakened, accounting for the reduced stability of R337H even under mildly acidic conditions. When this His is deprotonated, the interaction with D352 is also lost, accounting for the further destabilization observed under mildly alkaline conditions.
Simulations were carried out using both explicit and implicit solvent models, and both displayed similar behavior of the fluid salt-bridging cluster, suggesting that implicit solvent models can capture at least the qualitative features of this phenomenon as well as explicit solvent. Simulations under strongly acidic conditions in implicit solvent displayed the beginnings of the unfolding process, a destabilization of the hydrophobic dimer–dimer interface. Computational alanine scanning using the MM/PBSA method showed significant correlation to experimental unfolding data for charged and polar residues, but much weaker correlation for hydrophobic residues.
doi:10.1016/j.jmb.2007.07.080
PMCID: PMC2104543  PMID: 17900613
p53; MM/PBSA; Generalized Born; salt bridge; salt bridge exchange; molecular dynamics; protein flexibility; tetramerization domain; acid unfolding; destabilization; computational alanine scanning
25.  Hydrophobins are required for conidial hydrophobicity and plant root colonization in the fungal biocontrol agent Clonostachys rosea 
BMC Microbiology  2014;14:18.
Background
Filamentous fungi produce small cysteine rich surface active amphiphilic hydrophobins on the outer surface of cell walls that mediate interactions between the fungus and the environment. The role of hydrophobins in surface hydrophobicity, sporulation, fruit body formation, recognition and adhesion to host surface and virulence have been reported. The aim of the present study was to characterize the biological function of hydrophobins in the fungal biocontrol agent Clonostachys rosea in order to understand their potential roles in biocontrol mechanisms.
Results
Based on the presence of hydrophobin domains, cysteine spacing patterns and hydropathy plots, we identified three class II hydrophobin genes in C. rosea. Gene expression analysis showed basal expression of Hyd1, Hyd2 and Hyd3 in all conditions tested with the exception of induced Hyd1 expression in conidiating mycelium. Interestingly, up-regulation of Hyd1, Hyd2 and Hyd3 was found during C. rosea self interaction compared to interactions with the fungal plant pathogens Botrytis cinerea or Fusarium graminearum in dual culture assays. Phenotypic analysis of C. rosea deletion and complementation strains showed that Hyd1 and Hyd3 are jointly required for conidial hydrophobicity, although no difference in mycelia hydrophobicity was found between wild type (WT) and mutant strains. Interestingly, mutant strains showed increased growth rates, conidiation and enhanced tolerances of conidia to abiotic stresses. Antagonism tests using in vitro dual culture and detached leaf assays showed that the mutant strains were more aggressive towards B. cinerea, F. graminearum or Rhizoctonia solani, and that aggression was partly related to earlier conidial germination and enhanced tolerance of mutant strains to secreted fungal metabolites. Furthermore, in vitro Arabidopsis thaliana root colonization assays revealed reduced root colonization ability of the ΔHyd3 strain, but not for the ΔHyd1 strain. Furthermore, enhanced root colonization ability for the ΔHyd1ΔHyd3 strain was found in comparison to WT.
Conclusions
These results show a role for hydrophobins in conidial hydrophobicity, control of conidial germination under stress conditions, and in root colonization in C. rosea. However, functional studies of Hyd2 remains to be performed in order to fully assess the role of hydrophobins in C. rosea.
doi:10.1186/1471-2180-14-18
PMCID: PMC3922079  PMID: 24483277
Antagonism; Biocontrol; Clonostachys rosea; Filamentous fungi; Gene knockout; Hydrophobins

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