Abstract
Various recombinant light meromyosin (LMM) fragments were prepared from cDNAs encoding the 10°C and 30°C types of myosin heavy chain isoforms predominantly expressed in fast skeletal muscles of the 10°C-and 30°C-acclimated carp, respectively. These included three kinds of quarter fragments, 1/4-, 2/4-, and 4/4-quarter, composed of residues 1-130, 131-270, and 401-563 from the N-terminus, respectively, as well as three halves, N-, M-, and C-half fragments, containing residues 1-301, 131-400, and 302-563, respectively, and 69K fragments of residues 1-525. Unfortunately, in spite of extensive efforts, the 3/4-quarter fragment was not expressed for both 10°C and 30°C types in our expression system using Escherichia coli. All the LMM fragments except for the 10-and 30-2/4 quarters for the 10°C and 30°C types, respectively, exhibited a typical pattern of α-helix in CD spectrometry. When these were subjected to differential scanning calorimetry (DSC), 30°C-type LMM fragments were all found to be more thermostable than the 10°C-type counterparts. To identify amino acid substitutions responsible for different thermostabilities between the 10°C- and 30°C-type LMMs, six mutant proteins were prepared, mainly focusing on substitutions in the C-terminal half of LMM, and subjected to DSC and CD analyses. For three mutants in which two residues of the 10°C type were replaced by those of the 30°C type, 10-S355T/T361A, 10-M415L/L417V, and 10-S535A/ H536Q, the endothermic peaks in DSC increased by 1.4-2.0°C from that of the original 10°C type. The Tm values for two single-residue substitutions, 10-H449R and 10-T491I, shifted 0.8 and 1.3°C higher than that for the 10°C-type LMM, respectively, whereas the last mutant, 10-G61V, showed no change in thermostability. The finding that the difference in Tm values for major endothermic peaks from the 10-69K and 30-69K fragments was 4.6°C, which roughly corresponds to that between the original 10°C and 30°C types, suggested that the eight substitutions located in the C-terminal region of the 69K fragments (residues 302-525) are major candidates for the residues responsible for the difference in thermostability between the 10°C- and 30°C-type LMMs.
