Study On Molecular Engineering And Function Of Phycoerythrocyanin α-Subunit

Zhao discovered that in M.laminosus PecE/F is the regiospecific addition of phycocyanobilin (PCB) to Cys-84 of the phycoerythrocyaninα-subunit (PecA), and transform PCB to PVB. The classic photochromism of phycoerythrocyaninα-subunit involves the reversible shift of the visible absorption maximum when they are irradiated with light of certain wavelength. Its basis is the structure variation of the chromophore and the apoprotein.To study the spectral properties between wild type and mutant,on the basis of homology comparison and analysis of protein secondary structure, four mutants: PecA(C98I),PecA(Y65S),PecA(K83L),PecA(R93S) were successfully cloned by TaKaRa MutanBEST Kit.PCDFDuet-pecA was overexpressed in E. coli, and could, in vivo, attach PCB to pCOLADuet-pecE-pecF. Four forms of PVB-PecA were obtained: All mutants had highly reversibly photochromic activity. Compared to PVB-PecA, the photochemical activity of PVB-PecA(C98I) was just half; The spectrum of PVB-PecA(R93S) and PVB-PecA(Y65S) were the same as PVB-PecA, but the photochemical activity of PVB-PecA(R93S) was 75% to the nativeα-PEC. For PVB-PecA(K83L), the photochemical activity was raised to 102%, and fluorescence quantum yield was 2.49 times of the natural pigment. The spectral has been greatly improved, and provided a reference for the further molecular modification.