Kakinuma M, Hatanaka A, Fukushima H, Nakaya M, Maeda K, Doi Y, Ooi T, Watabe S
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan.
J Biochem. 2000 Jul;128(1):11-20. doi: 10.1093/oxfordjournals.jbchem.a022720.
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.
分别从编码主要在10℃和30℃驯化鲤鱼的快速骨骼肌中表达的10℃型和30℃型肌球蛋白重链亚型的cDNA制备了各种重组轻酶解肌球蛋白（LMM）片段。这些片段包括三种四分之一片段，即1/4 -、2/4 -和4/4 -四分之一片段，分别由N端的1 - 130、131 - 270和401 - 563位残基组成，还有三种半片段，即N -、M -和C -半片段，分别包含1 - 301、131 - 400和302 - 563位残基，以及1 - 525位残基的69K片段。遗憾的是，尽管付出了巨大努力，但在我们使用大肠杆菌的表达系统中，10℃型和30℃型的3/4 -四分之一片段均未表达。除了10℃型和30℃型分别对应的10 - 2/4和30 - 2/4四分之一片段外，所有LMM片段在圆二色光谱法中均呈现出典型的α -螺旋模式。当对这些片段进行差示扫描量热法（DSC）分析时，发现所有30℃型LMM片段的热稳定性均高于10℃型对应片段。为了确定导致10℃型和30℃型LMM热稳定性差异的氨基酸取代，制备了六种突变蛋白，主要集中在LMM C端区域的取代，并进行了DSC和圆二色分析。对于三个将10℃型的两个残基替换为30℃型残基的突变体，即10 - S355T/T361A、10 - M415L/L417V和10 - S535A/H536Q，DSC中的吸热峰比原始10℃型升高了1.4 - 2.0℃。两个单残基取代突变体10 - H449R和10 - T491I对应的熔解温度（Tm）值分别比10℃型LMM高0.8℃和1.3℃，而最后一个突变体10 - G61V的热稳定性没有变化。10 - 69K和30 - 69K片段主要吸热峰的Tm值差异为4.6℃，这大致与原始10℃型和30℃型之间的差异相当，这表明位于69K片段C端区域（302 - 525位残基）的八个取代是导致10℃型和30℃型LMM热稳定性差异的主要候选残基。
