Team:Groningen/Hydrophobins

Strongly hydrophobic proteins

In order to provide our Bacillus subtilis biofilm with hydrophobic properties we have turned to highly hydrophobic proteins originating from Streptomyces coelicolor, a soil dwelling bacterium which undergoes morphological differentiation as it goes through different stages in its life cycle, greatly resembling fungal growth. After submerged, vegetative growth aerial hyphae are formed which protrude from the moist substrate. The formation of these aerial hyphae appear to require strongly hydrophobic proteins called chaplins, which have previously been described by Claessen et al (2003) and Elliot et al.

Two subgroups

Chaplins can be categorized into two groups. The first group consists of chaplins A to C and are about 225 amino acids in size. These large chaplins contain a signal sequence, two hydrophobic chaplin domains, a hydrophilic region and a cell wall anchor. The second group includes chaplin D to H and are with around 63 amino acids smaller than the afore mentioned chaplins. Being smaller, they only contain a signal sequence followed by a hydrophobic chaplin domain.

Chaplins have the intrinsic property of assembling into rod-like structures called amyloid fibers. These fibers are very rigid and hard to break down and evern resist boiling in SDS which denatures almost all natural occuring proteins. They share distinguishing features with the medically important amyloid fibers that are characteristic for many neurodegenerative diseases such as Alzheimer's, Huntington's, systemic amyloidosis and the prion diseases.

Claessen, D; Rink, R; de Jong, W et al. 2003. A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils. Genes Dev 17 1714-1726

Physical properties

Interestingly, purified chaplins can be used to coat normally hydrophobic surfaces such as petri dishes, rendering them hydrophilic. This is due to their amphipatic nature, being hydrophobic on one side and hydrophilic on the other. In nature though they only coat the outside of the aerial hyphae of S. coelicolor hydrophobically. We pose that the assembly on the outside of cells is important for the amyloid fibers to polymerize into the right configuration to obtain extreme hydrophobicity. This is one of the reasons we chose to express chaplins in a biofilm as opposed to coat surfaces with purified chaplins.

Besides hydrophobicity and hydrophilicity we came across even another very interesting property. When in monomeric form purified chaplins appear to have very good oil dispersing properties. When in emulsion with water in oil, chaplins will interact with both water and oil dispersing the oil into the water.

Moreover, chaplins are extremely stable, both thermally and chemically. As an illustration, to purify them one has to turn to severe techniques like boiling in SDS and extraction with trifluoroacetic acid. Also, along the entire duration of our project – more than half a year – we did not observe any decline in the physical properties of our purified chaplins, being able to re-use the proteins over and over again.

Overall these small proteins appear to have great potential in all kinds of applicational fields, making them very interesting additions to the parts registry as biobricks. However it was their most remarkable property – their hydrophobicity – which drew our attention to them to use them in our project.