Abstract:

Neuroglobin(NGB), a newly discovered member of the hemoglobin superfamily, display a hexacoordination His-Fe-His in the absence of external ligands. In order to investigate the role of phenyla1anines(F) near bishistidyl ligands, the two neuroglobin mutants(F28Y, F106Y) were constructed, expressed, purified and characterized. The electrospray ionization mass spectroscopy results showed that the molecular weight of the two mutants correspond with the theoretical values. The UV-visible spectra of oxidized and reduced F28Y and F106Y were in agreement with the wild NGB, but the oxidized F28Y′s Soret band had a 2 nm blue shift, which suggested that the two mutants still remain hexa-coordinated form. The obvious red shift of F28Y′s fluorescence emission peak(F28Y: 340 nm→347 nm) relative to the wild NGB indicated that the mutant protein′s fluorophores exposed to more polar environment. The circular dichroism spectra results showed that the percentage of α-helix in F28Y, F106Y decreased and the β-sheet occurred in F28Y, this is caused by the specific site of F28 which sites at the outside of hydrophobic pocket and near the solvent surface. Thermal stability is NGB>F28Y>F106Y, which reflects that F106Y is more unstable. This may be explained on the basis of the strong hydrophobic interaction between F106 and heme, and also the decreased interaction, enables the heme disassociate from the protein chain more easily.

Key words: Neuroglobin; Mutant; Characterization; Stability