Ueki Nobuhiko, Ochiai Yoshihiro
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. 2006 Nov;140(5):649-56. doi: 10.1093/jb/mvj192. Epub 2006 Sep 20.
Structural stabilities of myoglobin (Mb) from several fish (scombridae) species differ significantly, although their amino acid sequence identity is very high (>95%), suggesting that only a few substitutions greatly affect the stability of Mb. Accordingly, recombinant Mbs with point mutation(s) derived from bigeye tuna Mb cDNA were expressed as GST-fusion proteins in the soluble fractions of Escherichia coli. After removal of the GST segment, the stability of five mutants, namely, P13A, I21M, V57I, A62G, and I21M/V57I, together with the wild type (WT) were investigated, taking temperature dependency of alpha-helical content and denaturant concentration dependency of Soret band absorbance as parameters. As a result, the stability of P13A against denaturants and its alpha-helical content at 10 degrees C was found to be the highest among the mutants, whereas those of A62G were the lowest. The stabilities of V57I and I21M/V57I were higher than that of WT, though that of I21M was nearly the same as WT. These findings suggest that the structural stability of fish Mb is tuned up only by the substitutions of a few amino acid residues located in the alpha-helical segments forming the hydrophobic heme pocket.
几种鲭科鱼类的肌红蛋白（Mb）的结构稳定性存在显著差异，尽管它们的氨基酸序列同一性非常高（>95%），这表明只有少数几个氨基酸替换会极大地影响Mb的稳定性。因此，将源自大眼金枪鱼Mb cDNA的点突变重组Mb作为GST融合蛋白在大肠杆菌的可溶性组分中表达。去除GST片段后，以α-螺旋含量的温度依赖性和Soret带吸光度的变性剂浓度依赖性为参数，研究了五个突变体（即P13A、I21M、V57I、A62G和I21M/V57I）以及野生型（WT）的稳定性。结果发现，在突变体中，P13A对变性剂的稳定性及其在10℃时的α-螺旋含量最高，而A62G的则最低。V57I和I21M/V57I的稳定性高于WT，不过I21M的稳定性与WT几乎相同。这些发现表明，鱼类Mb的结构稳定性仅通过位于形成疏水血红素口袋的α-螺旋片段中的少数几个氨基酸残基的替换来调节。
