Ochiai Yoshihiro, Ueki Nobuhiko, Watabe Shugo
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.
Comp Biochem Physiol B Biochem Mol Biol. 2009 Jun;153(2):223-8. doi: 10.1016/j.cbpb.2009.03.001. Epub 2009 Mar 11.
Structural stabilities of myoglobin (Mb) from several tuna fish species significantly differ from each other, although the amino acid sequence identities are very high (>95%), suggesting that limited number of substitutions greatly affect the stability of Mb. To address this hypothesis, attempts were made to elaborate recombinant tuna Mbs with point mutations on the different residues among fish Mbs. The expression plasmid constructs were based on bigeye tuna Mb cDNA sequence, and the recombinant proteins were expressed as GST-fusion proteins in Escherichia coli. After removal of the GST segment and affinity purification, the stability of five Mb mutants, namely, A49G, T91K, K92Q, V108A, and H112Q, together with the wild type (WT) were measured, taking temperature dependency of alpha-helical content and denaturant (urea and guanidine-HCl) concentration dependency of Soret band absorbance as parameters. As a result, the mutant H112Q showed much higher stability than WT, while the structures of K92Q, T91K and A49G mutants were destabilized. No essential change in helical content was observed for V108A, but the mutant was found to be destabilized easier by the denaturants. These findings suggested that the highly conserved residues among tuna species are responsible for their stability of Mbs, but a few non-conserved residues dramatically give rise to the differences in stability of Mbs among species.
几种金枪鱼物种的肌红蛋白（Mb）的结构稳定性彼此存在显著差异，尽管它们的氨基酸序列同一性非常高（>95%），这表明有限数量的取代会极大地影响Mb的稳定性。为了验证这一假设，研究人员尝试构建在鱼类Mb不同残基上具有点突变的重组金枪鱼Mb。表达质粒构建体基于大眼金枪鱼Mb cDNA序列，重组蛋白在大肠杆菌中作为GST融合蛋白表达。去除GST片段并进行亲和纯化后，以α-螺旋含量的温度依赖性和Soret带吸光度的变性剂（尿素和盐酸胍）浓度依赖性为参数，测定了五个Mb突变体（即A49G、T91K、K92Q、V108A和H112Q）以及野生型（WT）的稳定性。结果显示，突变体H112Q的稳定性远高于WT，而K92Q、T91K和A49G突变体的结构则不稳定。V108A的螺旋含量没有明显变化，但该突变体更容易被变性剂破坏稳定性。这些发现表明，金枪鱼物种中高度保守的残基决定了它们Mb的稳定性，但少数非保守残基显著导致了物种间Mb稳定性的差异。
