The class I hydrophobin EAS is part of a family of small, amphiphilic fungal proteins best known for their ability to self-assemble into stable monolayers that modify the hydrophobicity of a surface to facilitate further microbial growth. These proteins have attracted increasing attention for industrial and biomedical applications, with the aim of designing surfaces that have the potential to maintain their clean state by resisting non-specific protein binding. To gain a better understanding of this process, we have employed all-atom molecular dynamics to study initial stages of the spontaneous adsorption of monomeric EAS hydrophobin on fully hydroxylated silica, a commonly used industrial and biomedical substrate. Particular interest has been paid to the Cys3-Cys4 loop, which has been shown to exhibit disruptive behavior in solution, and the Cys7-Cys8 loop, which is believed to be involved in the aggregation of EAS hydrophobin at interfaces. Specific and water mediated interactions with the surface were also analyzed. We have identified two possible binding motifs, one which allows unfolding of the Cys7-Cys8 loop due to the surfactant-like behavior of the Cys3-Cys4 loop, and another which has limited unfolding due to the Cys3-Cys4 loop remaining disordered in solution. We have also identified intermittent interactions with water which mediate the protein adsorption to the surface, as well as longer lasting interactions which control the diffusion of water around the adsorption site. These results have shown that EAS behaves in a similar way at the air-water and surface-water interfaces, and have also highlighted the need for hydrophilic ligand functionalization of the silica surface in order to prevent the adsorption of EAS hydrophobin.

A lack in the detailed understanding of mechanisms through which proteins adsorb or are repelled at various solid/liquid interfaces limits the capacity to rationally design and produce more sophisticated surfaces with controlled protein adsorption in both biomedical and industrial settings. To date there are three main approaches to achieve anti biofouling efficacy, namely chemically adjusting the surface hydrophobicity and introducing various degrees of surface roughness, or a combination of both. More recently, surface nanostructuring has been shown to have an effect on protein adsorption. However, the current resolution of experimental techniques makes it difficult to investigate these three phase systems at the molecular level. In this molecular dynamics study we explore in all-atom detail the adsorption process of one of the most surface active proteins, EAS hydrophobin, known for its versatile ability to self-assemble on both hydrophobic and hydrophilic surfaces forming stable monolayers that facilitate further biofilm growth. We model the adsorption of this protein on organic ligand protected silica surfaces with varying degrees of chemical heterogeneity and roughness, including fully homogenous hydrophobic and hydrophilic surfaces for comparison. We present a detailed characterisation of the functionalised surface structure and dynamics for each of these systems, and the effect the ligands have on interfacial water, the adsorption process and conformational rearrangements of the protein. Results suggest that the ligand arrangement that produces the highest hydrophilic chain mobility and the lack of significant hydrophobic patches shows the most promising anti-fouling efficacy toward hydrophobin. However, the presence on the protein surface of a flexible loop with amphipathic character (the Cys3-Cys4 loop) is seen to facilitate EAS adsorption on all surfaces by enabling the protein to match the surface pattern.

Biofouling is a multibillion dollar problem in the modern world, stimulating a large research effort in developing antifouling surface coatings. Existing theories that attempt to explain underlying molecular mechanisms of biofilm formation and its attenuation are not consistent with experiments and focus on different aspects of the interactions. To address this knowledge gap, we report a computational molecular dynamics study in which we assess how chemistry and surface density of commonly used antifouling surface ligands affect the interfacial properties relevant to biofouling. We compare the hydration behavior and chain dynamics of poly­(ethylene glycol) (PEG) and poly­(2-oxazoline) (POX) modified silica surfaces as a function of chemical composition and grafting density. We show that PEG systems exhibit greater chain dynamics, while POX systems show superior hydropathicity and hydration behavior. The observed structure–property relations for the PEG- and POX-modified surfaces provide an improved understanding of the effects of molecular features on antifouling properties and highlight the importance of ligand mobility and interfacial water structure and dynamics for antifouling efficacy. The findings can be exploited in the rational design of biofouling-resistant surfaces for industrial and biomedical applications.

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