Structural characterization of the protein pheromones from the psychrophilic and early branching Euplotes species, E. petzi

Among the species of Euplotes that we are studying for the biology of their water-borne signaling protein pheromones, we have recently focused particular interest on E. petzi for two major reasons. It is a cold-loving (psychrophilic) species dwelling in the freezing Antarctic and Arctic sea waters, and forms, together with E. sinicus, the earliest branch of the Euplotes phylogentic tree. Using cultures of genetically distinct E. petzi strains, we have isolated and structurally characterized a family of four pheromones. With respect to the pheromone families known from E. raikovi, E. octocarinatus, E. nobilii and E. crassus, the E. petzi pheromones have the smallest dimensions of only 32 amino acids and the highest density of Cys residues lying in strictly conserved positions and predicted to form four intra-chain disulfide bridges. Although this high density of disulfide bridges would intuitively imply a quite compact globular molecular structure of these molecules, the nuclear magnetic resonance (NMR) determination of the solution structures of the two E. petzi pheromones designated “Ep-1” and “Ep-2” challenges this rationale. This determination shows that the molecular fold of E. petzi pheromones is characterized by a single extended alpha-helix, and by a spatial predominance of regions devoid of regular secondary organization. Considering that a three-helix fold and comparatively reduced extensions of un-structured regions are distinctive traits of the pheromone structures of Euplotes species which live in temperate waters and branch later than E. petzi in the Euplotes phylogenetic tree, our findings suggest two conclusions. The first one, of phylogenetic nature, is that the Euplotes pheromone structure evolves by increasing in dimensions and complexity. It appears to be further supported by the finding that also the macronuclear E. petzi pheromone genes have markedly reduced dimensions and a much simpler organization than their omologs of other Euplotes species. The second one, of adaptive nature, is that protein cold-adaptation is functionally correlated with an increased flexibility of the molecular backbone determined by an extension of unstructured regions. This conclusion is currently verified by analyzing the unfolding and refolding properties of E. petzi pheromones when exposed to increased temperatures and variations of other environmental parameters.