Cho-Fat Hu's Lab｜Institute of Cellular and Organismic Biology, Academia Sinica

Structure-function Relationship of Proteins that Function at Ambient, Low to Subzero Temperatures

Aquatic temperature is the major factor influencing poikilothermic (i.e., with variable body temperature) animals such as fishes. In general, body temperatures of fish depart little from ambient water temperatures. Therefore, fish need to regulate their metabolic rate to maintain energy requirements for survival at varying temperatures. Even though eurythermal fishes such as carp (Cyprinus carpio) do not have to confront temperature as low as -1.86 °C as do channichthyid icefishes of the Antarctic and the Arctic Seas, still they can survive temperatures as low as 12 °C. Therefore, it would be interesting to do a comparative study on differences in the primary, secondary, tertiary, and quaternary structures, biochemical kinetics, and molecular dynamics of metabolic enzymes of the common carp and icefishes in order to learn how these homologous enzymes function in such a broad range of temperatures.

We have begun our investigation by looking into the most primary and important process of energy homeostasis, and creatine kinase (CK; EC 2.7.3.2) is the first enzyme we have focused our attention on. CK enzymes catalyze the reversible phosphoryl transfer from phosphocreatine to ADP. In our previous studies, we cloned three different muscle-specific subisoforms of CKs from the common carp, designated M1-, M2-, and M3-CK. When specific activities were measured at different temperatures, the M1- and M2-CK proteins were found to exhibit their highest specific activities at 37 °C. However, the highest specific activity of the M3-CK protein was at 25 °C. In our more-recent studies, at 30 °C, the in vitro transcription/translation technique coupled with a glutathione-Sepharose bead pull-down assay demonstrated that M1- and M2-CK, but not M3-CK, interacted to form their respective homodimers. At temperatures below 20 °C, M3-CK interacted strongly to form its homodimer. Since M3M3-CK maintains its enzyme activity in quite a broad range of temperatures (30~15 °C), these two characteristics may qualify it to be termed a ‘psychrotolerant’ enzyme. We then used a protein-modeling method to try to explain the thermal stability of M3M3-CK. The calculation of the solvent-accessible surface area (ASA) that is buried in the contact interfaces of the M1M1-CK and M3M3-CK dimers showed a 12% decrease in the M3M3-CK interface.

If these studies were to be repeated in icefishes and the results compared, we would then have a more-complete picture of the structures, biochemical kinetics, and molecular dynamics of these enzymes. Finally, hyperthermophilic, thermophilic, and psychrophilic enzymes have attracted great interest because they can serve as model system for use by biologists, chemists, and physicists interested in understanding enzyme evolution, molecular mechanisms for protein thermostability, thermal instability, and function. Their intrinsic stability, instability, and activity at high and low temperatures offer immense biotechnological advantages over mesophilic enzymes.