THERMAL UNFOLDING OF 3 ACCLIMATION TEMPERATURE-ASSOCIATED ISOFORMS OFCARP LIGHT-MEROMYOSIN EXPRESSED BY RECOMBINANT DNAS

Differential scanning calorimetry (DSC) was performed to investigate t hermodynamic properties of three carp fast skeletal light meromyosin ( LMM) isoforms expressed in Escherichia coli by recombinant DNAs. Three isoforms were the 10 degrees C-, intermediate-, and 30 degrees C-type LMM predominantly expressed in carp acclimated to 10, 20, and 3 degre es C. The isoforms expressed in E. coli by recombinant DNAs exhibited a typical pattern of alpha-helix in CD spectroscopy with two minima at 222 and 208 nm, Moreover, the three isoforms formed paracrystals typi cal of LMM, suggesting that expressed proteins retained intact structu ral properties. When the LMM isoforms were subjected to DSC analysis, the 10 degrees C and 30 degrees C types showed endotherms having trans ition temperatures (T-m) at 35.1 and 39.5 degrees C, respectively, whi ch are responsible for thermal unfolding of alpha-helix. The intermedi ate type exhibited two comparable endotherms with T-m values at 34.9 a nd 40.6 degrees C, implying that it has intermediate thermodynamic pro perties between those of 10 degrees C and 30 degrees C types. However, a chimeric LMM having the 10 degrees C and 30 degrees C type as N- an d C-terminal halves, respectively, showed the DSC pattern typical of t he whole 30 degrees C-type molecule. On the other hand, another chimer ic LMM composed of the N-terminal 30 degrees C type and C-terminal 10 degrees C type gave the pattern of the full 10 degrees C type. These r esults suggest that thermodynamic properties of the C-terminal half la rgely account for thermal unfolding of the whole molecule.