title: Effect of amino acid replacements on the structural stability of fish myoglobin.
abstract: 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.
authors.affiliation: Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Ueki
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ochiai
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000502:physiology*; D000818:Animals; D004355:Drug Stability; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000235:genetics*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000187:drug effects; D016415:Sequence Alignment; D013696:Temperature; D013816:Thermodynamics; D014413:Tuna / Q000235:genetics*
chemical_list: D009211:Myoglobin