Thermal unfolding of three acclimation temperature-associated isoforms of carp light meromyosin expressed by recombinant DNAs.
Differential scanning calorimetry (DSC) was performed to investigate thermodynamic properties of three carp fast skeletal light meromyosin (LMM) isoforms expressed in Escherichia coli by recombinant DNAs. Three isoforms were the 10 degreesC-, intermediate-, and 30 degreesC-type LMM predominantly expressed in carp acclimated to 10, 20, and 30 degreesC. 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 typical of LMM, suggesting that expressed proteins retained intact structural properties. When the LMM isoforms were subjected to DSC analysis, the 10 degreesC and 30 degreesC types showed endotherms having transition temperatures (Tm) at 35.1 and 39.5 degreesC, respectively, which are responsible for thermal unfolding of alpha-helix. The intermediate type exhibited two comparable endotherms with Tm values at 34.9 and 40.6 degreesC, implying that it has intermediate thermodynamic properties between those of 10 degreesC and 30 degreesC types. However, a chimeric LMM having the 10 degreesC and 30 degreesC type as N- and C-terminal halves, respectively, showed the DSC pattern typical of the whole 30 degreesC-type molecule. On the other hand, another chimeric LMM composed of the N-terminal 30 degreesC type and C-terminal 10 degreesC type gave the pattern of the full 10 degreesC type. These results suggest that thermodynamic properties of the C-terminal half largely account for thermal unfolding of the whole molecule.
Kakinuma
M
M
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan.
Nakaya
M
M
Hatanaka
A
A
Hirayama
Y
Y
Watabe
S
S
Maeda
K
K
Ooi
T
T
Suzuki
S
S
D000064:Acclimatization; D000595:Amino Acid Sequence; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002347:Carps; D002942:Circular Dichroism; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000737:chemistry; D015879:Myosin Subfragments / Q000096:biosynthesis / Q000737:chemistry* / Q000648:ultrastructure; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis; D013696:Temperature; D013816:Thermodynamics
D004274:DNA, Recombinant; D015879:Myosin Subfragments; D011994:Recombinant Proteins