Structure-Function Relationships in Hydrophobins: Probing the Role of Charged Side Chains

Abstract

e 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.

Production and characterization of stable foams with fine bubbles from solutions of hydrophobin HFBII and its mixtures with other proteins

“…Hydrophobins are proteins exclusively produced by filamentous fungi, including some mushrooms [21][22][23]. They are stable and relatively small protein molecules.…”

“…The structure of HFBII determined from crystallized samples [24] shows that it is a single-domain protein with dimensions of 24 × 27 × 30 Å. On the surface of HFBII molecule, a hydrophobic patch and a larger hydrophilic portion are exposed, which give the molecule the character of an amphiphile (Janus-like particle); for details see [21][22][23][24][25]. The hydrophobins are very hard to denature -their aqueous solutions have been heated to 90 °C without any sign of protein denaturing [21,25].…”

Modular Architecture of Protein Binding Units for Designing Properties of Cellulose Nanomaterials

“…It is well documented that HFB forms multimeric complexes of quite large size. [21] The drive towards multimers increases with concentration, that is, during the drying process, we can expect stronger complex formation compared to the wet gel state. [19] In the dry state, we can expect that hydrophobic interactions between the proteins would allow some measure of fluidity in the interactions.…”

Modular Architecture of Protein Binding Units for Designing Properties of Cellulose Nanomaterials

“…The decrease in yield strength was 21.5, 19, and 20 MPa, while the decrease in slope after the yield point was 0.59, 0.86, and 1.11 GPa. It is well documented that HFB forms multimeric complexes of quite large size 21. The drive towards multimers increases with concentration, that is, during the drying process, we can expect stronger complex formation compared to the wet gel state 19.…”