Structure-function relationships in hydrophobins: probing the role of charged side chains

Abstract

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.

Fig 1

Three-dimensional structure of Trichoderma reesei HHBI (Protein Data Bank accession number 2FZ6). Basic and acidic residues are annotated and colored blue and red, respectively. The protein binds to hydrophobic substrates through the hydrophobic patch (shown in green).

Fig 2

Amino acid sequence of GFP-HFBI fusion protein. From residue 1, enhanced GFP; from residue 241, Gly-Ser linker; from residue 253, TEV protease recognition site; from residue 261, T. reesei HFBI; from residue 335, StrepII affinity tag; and from residue 344, ER retention signal. The sites where mutations were introduced are shown in red.

Fig 3

Asymmetric flow field flow fractionation elution profile of GFP-HFBI detected by elution of fluorescent protein (F; 488/507 nm). The first maximum at about 15 min is the monomer, and the second maximum at 28 min corresponds to the oligomeric state.

Fig 4

Correlation of GFP-HFBI variant oligomerization to adsorption to a 1-hexanethiol-coated hydrophobic substrate (A) and variant association with a nonionic surfactant in an ATPS (B).

Fig 5

Mutations affecting oligomerization also affect the adhesion of hydrophobins to a hydrophobic surface. The results show that a greater stability of oligomers in solution leads to less binding to surfaces and a lower stability of oligomers in solution leads to more binding to surfaces.