Effects of point mutations on the structural stability of tuna myoglobins.
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.
Ochiai
Yoshihiro
Y
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan. aochiai@mail.ecc.u-tokyo.ac.jp
Ueki
Nobuhiko
N
Watabe
Shugo
S
D000595:Amino Acid Sequence; D000818:Animals; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics*; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014413:Tuna
D009211:Myoglobin; D011994:Recombinant Proteins