Viviani Vadim R, Joaquim da Silva Neto Antonio, Ohmiya Yoshihiro
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA.
Protein Eng Des Sel. 2004 Feb;17(2):113-7. doi: 10.1093/protein/gzh016. Epub 2004 Jan 12.
To find the regions having a major influence on the bioluminescence spectra of railroad worm luciferases, we constructed new chimeric luciferases switching the fragments from residues 1-219 and from 220-545 between Phrixotrix viviani (PxvGR; lambda(max) = 548 nm) green light-emitting luciferase and Phrixothrix hirtus (PxhRE; lambda(max) = 623 nm) red light-emitting luciferases. The emission spectrum (lambda(max) = 571 nm) and K(M) for luciferin in the chimera PxRE220GR (1-219, PxhRE; 220-545, PxvGR) suggested that the region above residue 220 of PxvGR had a major effect on the active site. However, switching the sequence between the residues 220-344 from PxvGR luciferase into PxhRE (PxREGRRE) luciferase resulted in red light emission (lambda(max) = 603 nm), indicating that the region 220-344 by itself does not determine the emission spectrum. Furthermore, the sequence before residue 220 of the green-emitting luciferase is incompatible for light emission with the sequence above residue 220 of PxhRE. These results suggest that the fragments before and after residue 220, which correspond to distinct subdomains, may fold differently in the green- and red-emitting luciferases, affecting the active site conformation.
为了找到对铁路蠕虫荧光素酶生物发光光谱有重大影响的区域，我们构建了新的嵌合荧光素酶，将来自维氏磷光叩头虫（PxvGR；最大发射波长λ(max)=548nm）的绿色发光荧光素酶和多毛磷光叩头虫（PxhRE；最大发射波长λ(max)=623nm）的红色发光荧光素酶中1-219位残基片段和220-545位残基片段进行了交换。嵌合体PxRE220GR（1-219，PxhRE；220-545，PxvGR）的发射光谱（最大发射波长λ(max)=571nm）和荧光素的米氏常数（K(M)）表明，PxvGR第220位残基以上的区域对活性位点有重大影响。然而，将PxvGR荧光素酶220-344位残基之间的序列切换到PxhRE（PxREGRRE）荧光素酶中会导致红色发光（最大发射波长λ(max)=603nm），这表明220-344位区域本身并不能决定发射光谱。此外，绿色发光荧光素酶第220位残基之前的序列与PxhRE第220位残基以上的序列在发光方面不相容。这些结果表明，与不同亚结构域相对应的第220位残基前后的片段，在绿色和红色发光荧光素酶中可能折叠方式不同，从而影响活性位点的构象。
