Thermal unfolding of three acclimation temperature-associated isoforms of carp light meromyosin expressed by recombinant DNAs.
Thermal unfolding of three acclimation temperature-associated isoforms of carp light meromyosin expressed by recombinant DNAs.
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重组 DNA 表达的鲤鱼轻肌球蛋白的三种与驯化温度相关的亚型的热解折叠。
DOI：
10.1021/bi972344g
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发表时间：
1998
期刊：
影响因子：
2.9
通讯作者：
S. Suzuki
中科院分区：
文献类型：
--
作者：
M. Kakinuma;M. Nakaya;A. Hatanaka;Y. Hirayama;S. Watabe;K. Maéda;T. Ooi;S. Suzuki
用差示扫描量热法(DSC)研究了重组DNA在大肠杆菌中表达的3种鲤鱼快速骨骼轻质肌球蛋白(LMM)亚型的热力学性质。三种亚型是10度C、中间和30度C-型LMM，主要在10、20和30度驯化的鲤鱼中表达。重组DNA在大肠杆菌中表达的异构体在圆二色谱上呈现典型的α-螺旋模式，在222和208 nm处有两个极小值。此外，这三种异构体形成了典型的LMM的准晶，这表明表达的蛋白质保持了完整的结构特性。当对LMM异构体进行DSC分析时，10℃和30℃C类型的吸热曲线的转变温度(Tm)分别为35.1℃和39.5℃，这是负责α-螺旋的热展开的。中间型表现出两个可比的吸热曲线，Tm值分别为34.9℃和40.6℃，表明它具有介于10℃和30℃之间的中间热力学性质。然而，分别具有10度C和30度C型作为N-末端和C-末端半部分的嵌合LMM显示出整个30度C-型分子典型的DSC图谱。另一方面，另一种由N端30度C型和C端10度C型组成的嵌合型LMM给出了完整的10度C型图案。这些结果表明，C-末端一半的热力学性质在很大程度上解释了整个分子的热展开。
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.