Molecular aspects of the adaptation to low temperature of functional protein in Antarctic poikilotherms

Aim: Physiological processes are impaired by low temperature. The aim of our research was: i) to understand the molecular aspects of the physiologic and phylogenetic adaptation of functional mechanisms of life to the cold, which allow poikilotherms to survive well in thermodynamically disadvantageous conditions; ii) to provide the scientific background for the biotechnological exploitation of cold-adapted functional mechanisms. Methods: Functional protein (such as carbonic anhydrase and the dipeptide carrier) were isolated from Antarctic teleosts, purified, sequenced and processed for the biochemical and the physiological characterisation. The analysis of the protein lipid microenvironment was performed to assess the role of the lipid-protein interactions on the overall functionality of the protein. FPLC and HPLC were used for protein isolation and purification. Sequencing was carried out by direct protein and cDNA sequencing and mass spectrometry. Isoelectric focusing and Western blot analysis were used to show post-translational modifications. Fluorimetry, spectrophotometry, isotope labelled substrates were used for the biochemical/physiological characterisation of enzymes and transporters. Gas-chromatography, TLC, fluorimetry and spectrophotometry allowed the analysis of the lipid microenvironment. Bioinformatics was applied to perform the phylogenetic analyses. Results: Some molecular aspects of the capability of protein to perform well at low temperature have been identified. The phylogenetic relationships with analogues from higher temperature have been assessed. The lipid analysis showed the adaptive contribution of the membrane lipid microenvironment to the protein adaptation to cold. Conclusions: The results obtained extend our knowledge about the key molecular characteristics and mechanisms which contrast the thermodynamic disadvantages of low temperature toward any physiological process.