Differential scanning calorimetry of light meromyosin fragments having various lengths of carp fast skeletal muscle isoforms.
Various recombinant light meromyosin (LMM) fragments were prepared from cDNAs encoding the 10 degrees C and 30 degrees C types of myosin heavy chain isoforms predominantly expressed in fast skeletal muscles of the 10 degrees C- and 30 degrees 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 degrees C and 30 degrees 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 degrees C and 30 degrees C types, respectively, exhibited a typical pattern of a-helix in CD spectrometry. When these were subjected to differential scanning calorimetry (DSC), 30 degrees C-type LMM fragments were all found to be more thermostable than the 10 degrees C-type counterparts. To identify amino acid substitutions responsible for different thermostabilities between the 10 degrees C- and 30 degrees 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 degrees C type were replaced by those of the 30 degrees C type, 10-S355T/T361A, 10-M415L/L417V, and 10-S535A/H536Q, the endothermic peaks in DSC increased by 1.4-2.0 degrees C from that of the original 10 degrees C type. The T(m) values for two single-residue substitutions, 10-H449R and 10-T491I, shifted 0.8 and 1.3 degrees C higher than that for the 10 degrees C-type LMM, respectively, whereas the last mutant, 10-G61V, showed no change in thermostability. The finding that the difference in T(m) values for major endothermic peaks from the 10-69K and 30-69K fragments was 4.6 degrees C, which roughly corresponds to that between the original 10 degrees C and 30 degrees 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 degrees C- and 30 degrees C-type LMMs.
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.
Hatanaka
A
A
Fukushima
H
H
Nakaya
M
M
Maeda
K
K
Doi
Y
Y
Ooi
T
T
Watabe
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002347:Carps; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D018656:Muscle Fibers, Fast-Twitch / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D015879:Myosin Subfragments / Q000737:chemistry* / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D017354:Point Mutation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017384:Sequence Deletion; D013816:Thermodynamics
D015879:Myosin Subfragments; D010446:Peptide Fragments; D020033:Protein Isoforms; D011994:Recombinant Proteins