Abstract
Three psychrophilic protein pheromones (En-1, En-2 and En-6) from the polar ciliate, Euplotes nobilii, and six mesophilic pheromones (Er-1, Er-2, Er-10, Er-11, Er-22 and Er-23) from the temperate-water sister species, Euplotes raikovi,were studied in aqueous solution for their thermal unfolding and refolding based on the temperature dependence of their circular dichroism (CD) spectra. The three psychrophilic proteins showed thermal unfolding with mid points in the temperature range 55–70 °C. In contrast, no unfolding was observed for any of the six mesophilic proteins and their regular secondary structures were maintained up to 95 °C. Possible causes of these differences are discussed based on comparisons of the NMR structures of the nine proteins.
Highlights
Protozoan ciliates represent a major micro-eukaryotic component of the polar ecosystem [1,2], which can readily be collected from every aquatic habitat for use in stable laboratory cultures [3].Strains of Euplotes species such as E. patella, E. raikovi, E. octocarinatus and E. crassus inhabiting non-polar temperate waters, and of E. nobilii inhabiting Arctic and Antarctic waters are capable of secreting cell type-specific signaling proteins genetically specified at a single multi-allelic locus [4,5]
For all the three psychrophilic pheromones En-1, En-2, and En-6 of E. nobilii, the circular dichroism (CD) spectra (Figure 2) show that the regular secondary structures are unfolded at 95 °C, and that this unfolding is reversible upon cooling of the solutions to the starting temperature at 20 °C
The psychrophilic and mesophilic pheromone families of the protozoan ciliate Euplotes studied here are both represented by single-domain small disulfide-rich proteins, which have usually been shown to be outstandingly stable with regard to thermal denaturation in aqueous solution [26±28]
Summary
Strains of Euplotes species such as E. patella, E. raikovi, E. octocarinatus and E. crassus inhabiting non-polar temperate waters, and of E. nobilii inhabiting Arctic and Antarctic waters are capable of secreting cell type-specific signaling proteins genetically specified at a single multi-allelic locus (designated as mating-type, or mat locus) [4,5]. In addition to the full-length coding gene sequences [8±10], the three-dimensional molecular structures of a significant number of pheromones were determined by NMR spectroscopy in solution, firstly, from the temperate-water species, E. raikovi [11±17], and subsequently from the polar-water species, E. nobilii [18±20] These mesophilic (E. raikovi) and psychrophilic (E. nobilii) pheromone families, both characterized by small, helical and disulfide-rich proteins of 37 to 63 amino acids, represent an interesting source of material for structure based comparative studies of protein adaptation to cold. Considering that NMR solution structures are available for all the nine proteins, and for one (i.e., Er-1) is available the crystallographic structure [21], we expect that these data will be of interest for in-depth studies of correlations between molecular protein structure, thermodynamic stability, and cold adaptation
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