Thermodynamic Stability of Psychrophilic and Mesophilic Pheromones of the Protozoan Ciliate Euplotes

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.


Introduction
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 (designated as mating-type, or mat locus) [4,5]. These waterassociated with the genetic mechanism of the mating types and act as prototypic autocrine (autologous) growth factors and as paracrine (heterologous) inducers of mating pair formation [6,7]. 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, thus represent an interesting source of material for structure based comparative studies of protein adaptation to cold.
Here we present data on the thermal denaturation of the three pheromones En-1, En-2 and En-6 from the psychrophilic pheromone family of E. nobilii, and the six pheromones Er-1, Er-2, Er-10, Er-11, Er-22 and Er-23 from the mesophilic pheromone family of E. raikovi ( Figure 1). Considering that NMR solution structures are available for all the nine proteins, and for one (i.e., Er-1) is available also 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.

Materials and Methods
The isolation and purification of the nine proteins investigated in this paper has previously been described [22 25]. Lyophilized samples of each pheromone were dissolved in 20 mM sodium phosphate buffer at pH 6.0 and diluted to a protein concentration of 20 µM before aliquoting into a 0.1 cm path length quartz cuvette. CD experiments were recorded using the Temperature/Wavelength Scan software supplied with the Jasco 815 CD spectrophotometer. Melting curves over the range 20 95 °C were measured at a constant wavelength of 220 nm by increasing the temperature at a rate of 1.0 or 0.5 °C/min. Wavelength scans from 260 190 nm were measured at 5 °C intervals.
In additional exploratory studies, the chemical denaturants guanidine-HCl and urea were added to solutions of the mesophilic pheromones Er-1, Er-10, Er-22 and Er-23 to further test their stabilities. In particular melting curves were recorded for Er-1 in 7.8 M urea and 20 mM sodium phosphate at pH 6.0, 6 M guanidine-HCl and 20 mM sodium phosphate at pH 6.0, 4 M guanidine-HCl and 20 mM sodium phosphate at pH 6.0, 4.5 and 3.0, and 6 M urea at pH 3.0. Similar analyses on Er-10, Er-22 and Er-23 were performed using 4 M guanidine-HCl in 20 mM formic acid at pH 2.0.

Results
For all the three psychrophilic pheromones En-1, En-2, and En-6 of E. nobilii, the 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. Nevertheless, among the individual proteins there are appreciable variations with regard to the shape of the thermal unfolding curves. For En-6 a nearly symmetrical sigmoidal denaturation curve was observed with a midpoint near 65 °C, and a sigmoidal curve was also obtained for the refolding upon cooling of the solution; in addition, only a very small loss of protein was recorded during the unfolding/refolding procedure. On the other hand, En-1 and En-2 showed more sluggish unfolding transitions, and for En-2 there was an indication that the unfolding and refolding processes involve equilibria between more than two states. For the present qualitative survey of the stability of these three proteins we retain that, similar to En-6, the regular secondary structures in En-1 and En-2 are unfolded at 95 °C and refolded upon cooling of the solutions to 20 °C.
In the family of the six mesophilic pheromones Er-1, Er-2, Er-10, Er-11, Er-22, and Er-23 from E. raikovi, the regular secondary structures manifested in the CD spectra at 20 °C were found to be maintained up to the highest temperature studied (Figure 2). The temperature dependence of the signal intensity at 220 nm did not provide evidence for unfolding, and for Er-22 the small reduction of the signal intensity at the higher temperatures appeared to be fully reversible upon solution cooling. The data recorded for this pheromone are representative of the observations made with the other pheromones Er-1, Er-2, Er-10, and Er-11. Also the pheromone Er-23 showed qualitatively similar behavior, but it was clearly the most stable member of the E. raikovi mesophilic protein family since it did not undergo denaturation (Figure 3).

Figure 2.
Temperature-induced denaturation of the E. nobilii psychrophilic pheromones En-6, En-1 and En-2 monitored by CD spectroscopy. (a) Temperature variation of the signal intensity at 220 nm during heating and cooling over the range from 20 °C to 95 °C. (b) CD spectra at different temperatures, as indicated by the color code in the figure. The protein concentration was 20 µM in 20 mM sodium phosphate at pH 6.0, and the cell length 0.1 cm. The temperature variation of the signal intensity at 220 nm in panels (a) was recorded at a rate of 1.0 °C/min. Scans were recorded with a speed of 100 nm/min, in 5 °C intervals over the temperature range of panels (a); for improved clarity, only four traces are shown in panels (b).

En-6
En-1 En-2 In view of the high thermal stability of the E. raikovi pheromones in neutral aqueous solution, we also performed exploratory experiments with the addition of chemical denaturants (see Materials and Methods). These experiments provided further indications of the remarkably high stability of these proteins. We did not observe their full unfolding in the temperature range 20 95 °C with the solution conditions listed in the Materials and Methods section, although partial melting within this temperature range was observed for some of them.

Discussion
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]. However, despite their extensive homology on the level of the amino acid sequences and three-dimensional structures, the psychrophilic E. nobilii pheromones showed a significantly lower thermal stability than their mesophilic E. raikovi counterparts. This finding coincides with observations derived from comparisons between other psychrophilic and mesophilic homologous proteins [29 33]. However, while these comparisons are essentially based on individual proteins from distantly related organisms, our finding involves families of proteins with known NMR solution structures [11 21] from two closely related species [34,35]. It therefore provides data which, at least in principle, are more reliable (being unaffected by the evolutionary noise which is intrinsic to comparisons between distantly related systems) for further detailed analyses by other groups of researchers who are interested in studying the correlations between protein structure, thermodynamic stability, and cold-adaptation.  Figure 2, except that all the spectra recorded in 5 °C temperature intervals are shown in panels (b).

Er-22
Er-23 As previously discussed [3,10,21], the two homologous protein families of E. nobilii psychrophilic pheromones and E. raikovi mesophilic pheromones differ significantly in the composition of polar, hydrophobic and aromatic amino acids, in global hydrophilicity and hydrophobicity, as well as in various aspects of their three-dimensional structures. It will now be interesting to explore the extent to which these physico-chemical and structural variations can be related to variations of the stability of the folded proteins. Two structural features distinctive of the two pheromone families appear to be particularly promising starting points for further investigations into the structural basis of the observed differences in the thermal stability of these proteins. One is the N-terminal elongation of 10 to 12 residues, which is common to all the E. nobilii pheromones and has no counterparts in any of the E. raikovi pheromones. It includes only a 3 10 -helical turn as regular secondary structure and is devoid of connections through disulfide bonds to other parts of the proteins. Secondly, there is a higher density of disulfide bonds in the mesophilic E. raikovi pheromones than in the psychrophilic ones of E. nobilii. On average, the E. raikovi pheromones contain one disulfide bond per 13 amino acid residues, while in the E. nobilii pheromones there is one disulfide bond per 15 residues. In particular, the high density of one disulfide bond per 10 residues appears to be the dominant feature that makes Er-23 most stable among the E. raikovi pheromones, notwithstanding that Er-23 mimics the E. nobilii psychrophilic pheromones with regard to the contents of polar and hydrophobic residues as well as the aliphatic index.

Conclusions
The key observation reported in this paper is that the E. nobilii and E. raikovi pheromone families show a thermal denaturation behavior that is uniform among their members and significantly divergent between them. The mesophilic E. raikovi pheromones have higher stability by at least 30 °C when compared to the psychrophilic E. nobilii pheromones. Given the small size of these water-borne signaling proteins and the availability of their three-dimensional structures, the data presented in this communication should be of interest for systematic investigations of protein adaptation to cold environments.