Variability of Paralithodes camtschaticus hemocyanin isoforms during the molting cycle: Quaternary structure and...

The multifunctionality of hemocyanins is intrinsically linked to their molecular heterogeneity. The expression levels of different hemocyanin subunits can change in response to environmental influences and during ontogenetic processes. In this work, we identified a number of differentially expressed isoforms of hemocyanin from the hemolymph of red king crab Paralithodes camtschaticus of the Western Sakhalin population at different stages of the molting cycle using proteomics informed by transcriptomics. Determination of the quaternary structure of hemocyanin at different stages of the molting cycle using size-exclusion chromatography and dynamic light scattering showed that the variability of the subunit composition did not significantly affect the oligomerization propensity of the protein. Differential scanning calorimetry was performed with subunits and heterohexamers of hemocyanin at different stages of the molting cycle. Native heterohexamers showed a significant similarity in their thermal denaturation behavior which could be interpreted in terms of a two-state model. Based on the bioinformatics-assisted analysis of hemocyanin isoforms sequences we assume that the rate-limiting step in the denaturation process of the native heterohexamers is the unfolding of cooperative thermodynamic domains formed through interactions between highly conserved residues from the central domains of the subunits. Thus, the variability in the isoform composition of native oligomers of Paralithodes camtschaticus hemocyanin during the molting cycle, does not significantly affect their quaternary structure or conformational stability due to the features of their molecular architecture.