Hydrophobins of this class have between 85 and 95 amino acid residues. The length of its Cys3–Cys4 loops varies between 4 and 44 residues, while its Cys4–Cys5 loops vary between 8 and 23 residues (Figure 2) [14]. The monolayers generated by this class are robust and resistant to enzymatic treatments, detergents such as SDS (2%), and high temperatures (100°C). However, they are dissociable with strong acid treatments (formic acid and trifluoroacetic acid).

An example of this class is hydrophobin SC3, from the basidiomycete S. commune, which reduces the surface tension of water from 72 to 24 mJ/m² at 25°C [2]. These proteins are the most studied within the hydrophobin family since they were among the first to be discovered and described. SC3 has 112 amino acids and weighs 13,431 Da (UniProt ID: P16933 SC3_SCHCO). In early experiments carried out by [15], it was discovered that the four disulfide bridges present in all hydrophobins, regardless of their classification, are essential for protein stability in their monomeric form since they prevent premature self-assembly (that is, without being in contact with the interface). In addition to these disulfide bridges, SC3 contains between 17 and 22 mannose residues linked to threonine residues in the N-terminal part of the protein, that is, exposed, which gives its characteristic properties to the hydrophilic side of the monolayer. To produce amphipathic monolayers, SC3 undergoes conformational changes within an oligomerization process, requiring a critical concentration of 50 μg/mL [2].

The first conformational change involves going from the monomeric configuration of the protein (the one that maintains a high solubility in aqueous media and is secreted by the cell) to the α-helical structure. This change occurs when the monomers are in contact with the hydrophilic–hydrophobic interface; such contact causes the hydrophobin SC3 to proceed into an intermediate stage, where the content of the α-helix structure increases, allowing hydrophobins to form amyloid fibers, that is, an intermediate between the monomeric and the β-folded conformation [13]. Therefore, it is considered that the change from the monomer state to a helical intermediate is responsible for initiating polymerization. Similarly, the second conformational change consists of the lateral association of β-sheet species to generate rods or fibers of 10 nm in diameter (each composed of 2–3 protofilaments of 2.5-nm wide) and monolayers on a surface or substrate [16]. In addition, Scholtmeijer et al. reported that specific components of the cell wall, such as schizophyllan and β-1-3 glucan, accelerate the formation of β-sheets. The latter was established after observing an increase (80%) in thioflavin T (ThT) fluorescence in 16 h when schizophyllan was added to the solution; in its absence, only a 25% increase in fluorescence was observed. However, the exact mechanism by which they promote agglutination is barely known [17].

EAS is another example of a class I hydrophobin secreted from Neurospora crassa. Its function is to facilitate the formation and dispersal of spores and the formation of impermeable amphipathic monolayers on them. This monolayer is made up of rods associated laterally, which optimizes the dispersion of spores in air and water, thus favoring the survival of N. crassa. It has been established that its Cys7–Cys8 loop spans from residues 19 to 45, constituting the largest loop, giving this region a crucial role in the protein aggregation propensity [18].

Monolayers generated by class I hydrophobins could be detected with fluorescence assays using ThT and staining with Congo red (CR) dye. Implementing these techniques aims, through agitation, to create hydrophobic–hydrophilic interfaces that promote intermolecular association (polymerization) and the consequent formation of amyloid fibers. The amphipathic nuclear structure (present in all these proteins despite the considerable sequence variation in the family) determines the unique properties of hydrophobins. Regardless of the differences between classes I and II, they have no distinction regarding their life cycle [18].

Hydrophobins of this class have approximately 70 residues. The length of its Cys3–Cys4 and Cys7–Cys8 loops is preserved [18]. Class II hydrophobins are abundantly secreted into the growth medium and the cell surface. Monolayers generated are not amyloid in nature as they dissociate into 70% ethanol, detergents such as SDS (2%), and high temperatures (100°C). Furthermore, they are not resistant to acid or enzymatic treatments [2]. An example of this class is the hydrophobin cerato-ulmin (CU) obtained from Ophiostoma ulmi, isolated and partially characterized approximately 25 years ago. CU self-assembles in stirred aqueous solutions subjected to vacuum or aeration, and in large quantities, it forms fibrils that increase the turbidity of the solution and membranes on the surface of an aqueous solution [19].

Class I and II hydrophobins differ in their arrangement of amino acids, that is, while the loops formed by disulfide bridges vary in the number and type of amino acids among class I hydrophobins, the loops of class II hydrophobins are more conserved [2] (Figure 3).

Another significant difference between both classes is that in class I, the charged residues are delimited to a section within the three-dimensional conformation of the proteins, that is, the charge of the protein is localized. In contrast, the charged residues in class II hydrophobins are better distributed throughout the three-dimensional structure (Figure 4). This difference is responsible for the distinction between a hydrophobic and hydrophilic side in class I proteins and the absence of this distinction in class II hydrophobins. However, in the latter, hydrophobic regions could also be exposed to the phase gas from a hydrophobic–hydrophilic interface to form the characteristic monolayers of these proteins. This quality explains the ability of hydrophobins to form amphipathic monolayers in both classes [18].

Hydrophobins play a vital role in the growth and development of most filamentous fungi. The formation of aerial hyphae is a process in which the secretion of surfactant molecules reduces surface tension, affecting the cohesive force between water molecules on the surface of aqueous environments (Figures 5A, B) [20, 21]. These surfactant molecules are hydrophobins, which are excreted to form a layer or rodlet around the hyphae, conferring hydrophobicity to the surface of the fungus. Self-assembly of hydrophobins on the cell wall surface gives an amphipathic trait to aerial hyphae (Figure 5C), fruiting bodies, the lining of air cavities in fruiting bodies (Figure 5D), and spores. The preceding results in the attachment of hyphae to hydrophobic surfaces. Class I hydrophobins assemble into a stable, amyloid-type fibril that can only be dissociated using trifluoroacetic and formic acid. In contrast, class II hydrophobins do not assemble into amyloid-type fibrils and can dissociate in 70% ethanol, 2% SDS, or by applying pressure [21, 22].

The first step in forming fungal aerial structures is the escape of individual hyphae from the moist substrate into air. Hyphae secrete hydrophobins, which remain free in the aqueous medium and, therefore, cannot diffuse, remaining at the cell wall–air interface. This process induces the formation of a film of these amphipathic proteins, conferring hydrophobicity to the fungal hyphae. On one hand, the hydrophilic part faces the hydrophilic cell wall, while the hydrophobic part is exposed [20, 22].

The hydrophobic part of hydrophobins has a characteristic rodlet pattern, which is observed in layers on the surfaces of fungal aerial structures. As Wosten suggests, these layers could result from assembling class I hydrophobins [22].

The dikaryotic phase involves coating air channels within the fruiting bodies. This maintains the hydrophobic nature of air channels and avoids waterlogging, thus allowing the aeration of multicellular structures. The last phenomenon was observed in the hydrophobins SC4 and ABH1 produced by S. commune and A. bisporus, respectively [23].

Hydrophobins in an aqueous solution can self-assemble, at the interfaces, into an amphipathic film, reversing the wettability of surfaces and, thus, changing a hydrophilic surface to a hydrophobic surface and vice versa. A hydrophilic surface has a contact angle of less than 90°, while a hydrophobic surface shows a contact angle between 90° and 120° (Figure 6) [2]. Hydrophobin surface activity has been measured by the contact angle of water droplets on different surfaces, such as Teflon, glass, mica, and polyethylene [24].

Hydrophobins facilitate adhesion to the insect cuticle for invasion. The infection begins with the attachment of the spore to the insect cuticle, and then, enzymes, proteins, and other factors are expressed to facilitate the penetration of the cuticle [25, 26]. It has been reported that hydrophobins from Aspergillus fumigatus contribute to the immune evasion of conidia by hiding host Dectin-1- and Dectin-2-dependent immune recognition of fungal spores [27].

Many conidiospores are characterized by having a rodlet surface made up of class I hydrophobins, while class II hydrophobins are present in yeast-type cells. In addition to aiding the dispersion of contagious propagules by wind, these hydrophobin layers also serve as a scaffold for the attachment to the host surface. Once the infectious propagule attaches, it can colonize the host, which may be mediated by forming an infection structure called the appressorium [22, 25].

In the food industry, foams constitute many products manufactured and sold today. These foams range from the foam of a beer to the porous structure of bread. Hydrophobins are ideal as a case study for foams and emulsions over other small proteins used to stabilize emulsions and foams due to their high surfactant nature and self-assembling properties [28]. These proteins present significant structural variability, generating even more tremendous potential as new surfactant molecules. Hydrophobins bind together, producing an elastic membrane at the interface that produces a barrier against the escape of air particles. These small proteins reduce the surface tension and cover the surface quickly, allowing smaller air cells to be created, thus producing a buffer against coalescence [29].

Amyloid proteins are biomolecules that result from unconventional folding and are characterized by their ability to self-assemble through hydrogen bonds into fibrous-type quaternary structures. These aberrant proteins are involved in diseases known as amyloidosis, such as Alzheimer’s disease, Parkinson’s disease, cystic fibrosis, type II diabetes, and Huntington’s disease, which have no cure [30, 31]. Functional amyloids are considered double-edged swords since, due to this, they have gained the interest of many scientists to try to understand the conditions and the mechanism that allow the transformation of normal proteins into toxic species capable of causing diseases so severe that they can lead to death. To make this possible, scientific research is dedicated to proposing and consolidating biological models that can adequately represent diseases, making it viable to extrapolate behavior from the model to human physiology. Those structures require amyloid conformation to carry out their biological function (which does not derive from unconventional protein folding). There are multiple examples of functional amyloids in nature, ranging from yeasts, bacteria of various genera (both pathogenic and non-pathogenic), protozoans, plants, and even mammals [14].

The self-assembly of insoluble proteins as aggregates or deposits can be toxic and harmful to any organism. However, hydrophobins represent the duality of amyloids since their study allows the generation of knowledge about organisms that use non-traditional proteins to carry out their biological functions. In contrast, in other situations, these proteins imply incurable diseases [30].

Hydrophobins offer a simple and efficient alternative to optimize the solubility and stability of medications in suspension. Solubility and stability are factors directly related to the availability of the drug, which can be modified in terms of the concentration of the active molecule or the release time (when this is a treatment that requires the prolonged release of a drug). In addition to the above application, hydrophobins in the pharmaceutical industry protect the drug during processing, formulation, and storage, maintaining its properties under optimal conditions to provide a more effective treatment [37]. This application is useful when it comes to drugs administered orally due to the process that takes place before the drug reaches its active site [38].

Class II hydrophobins from Trichoderma reesei (HFBI and HFBII) were used to interact strongly with nonionic surfactants, thus optimizing the purification of recombinant proteins in biphasic aqueous systems [20]. [39] tested this application for the first time, obtaining a yield of 90% and a 6-fold increase in purification in the concentration of the purified protein. Another example within this category is the ability of hydrophobins to facilitate the overexpression of proteins from plant extracts. In this case, hydrophobins fuse with a marker to form protein bodies within the host cell, protecting external proteins from degradation due to host defense action, thus reducing necrosis in the leaves [40].

[41] proposed a methodology for coating biomedical silicone implants with titanium dioxide biofilms. This compound, with electrical, optical, and chemical properties, helps develop microelectronic devices, photonic materials, catalysts, and bioremediation and medical treatments among others. Biofilms were generated from a modified class I hydrophobin (derived from the DewA protein of the organism Aspergillus nidulans). The implant coating was uniform, resistant, and elastic, characteristics necessary for biocompatibility.

In another study, [42] modified the same hydrophobin mentioned above, inserting the RGD sequence, or the globular domain of laminin, to functionalize the surfaces of orthopedic titanium implants. This functionalization optimized the adhesion of human cells and impaired the adhesion of pathogens such as Staphylococcus aureus, thus minimizing the risk of bacterial infection.

Functionalization of plastic surfaces began with class I hydrophobins SC3 and SC4 (both in their native and recombinant forms) on the Teflon surface [2]. The interest in functionalizing these materials (plastics) appeared due to the hypothesis of biocompatibility that would allow cell adhesion and tissue regeneration. Subsequently, it was extended to antimicrobial functionalization with two types of hydrophobins (Vmh2 and Pac3), originating from the organism Acremonium sclerotigenum, where the reduction of biofilms formed by different strains of S. epidermidis on polystyrene surfaces was observed [43].

Regarding biosensors on plastic surfaces, the EAS (for “easily wettable”) hydrophobin from the organism N. crassa was used for the α-factor detection and quantification, a peptide hormone from yeast. The methodology consists of functionalizing a polystyrene surface on which an inverted enzyme-linked immunosorbent assay (ELISA) is performed [44].

Another application is pesticide detection using the hydrophobin Vmh2 from Pleurotus ostreatus. In this technique, Vmh2 is fused with the enzyme glutathione S-transferase to quantify pesticides such as molinate and captan, which inhibits its enzymatic activity. It is worth mentioning that this test is also carried out on a functionalized polystyrene surface [45].

The modification of plastic materials using hydrophobins has increased in recent years. Different plastics have been reported using class I hydrophobins from native and recombinant sources. Such is the case with Teflon, polystyrene, polycaprolactone, and plastic biliary stents, which have applications after being modified in the medical area, biosensors, and enzyme immobilization, respectively [46].

The 2D materials have a large surface area about their volume and offer the possibility of a wide variety of uses through their functionalization. These 2D materials have allowed the synthesis of high-quality, stable liquid dispersions, which consist of a few layers, maintaining an ideal thickness, for applications related to photoluminescence [47]. Carbon nanotubes (CNTs) are allotropes of carbon (such as diamond, graphite, and fullerenes); their structure is described as a sheet of graphene rolled to form a tube. This winding can be single or multiple, resulting in a single-walled tube or one with multiple walls. The biggest problem with a CNT is that its low solubility in water and toxicity limit its use in some biomedical issues [48]. Due to this, hydrophobins have been widely used to minimize these drawbacks and provide these CNTs with a plethora of applications, including biological, pharmaceutical, chemical, and industrial applications [49].

CNTs, 2D materials, and other materials could offer applications related to enzyme immobilization, optimization of reaction kinetics, and biocatalytic efficiency in industrial processes. For example, the recombinant hydrophobin HFB (HYDPt-1) from the organism Pisolithus tinctorius was used to immobilize small molecules of an electroactive nature on three different substrates [50]. The immobilization of molecules with hydrophobins allows the optimization of immunoassays since materials coated by these proteins are used as platforms in antibody capture systems. One of the materials that can be covered with hydrophobins, such as Vmh2, is glass. The functionalization of this surface allowed the creation of a platform for immobilizing nanomaterials, such as quantum dots and graphene oxides [51].

Due to the biodegradable and non-toxic properties of hydrophobins, they have been proposed as an ecological alternative in many commercial sectors, the textile industry being one of the most striking. Among these innovations is the modification of fabrics, making them more hydrophobic, functionalization with antimicrobial agents (such as zinc and silver oxide nanocomposites), and even ignition resistance. The advantage of using hydrophobins in the textile sector is that the coating of the fabrics does not change their feel, comfort, or weight, thus making them ideal green alternatives. Among the properties that hydrophobins can attribute to textiles when functionalized, the following stand out.