PEDS Advance Access originally published online on January 12, 2004
Protein Engineering Design and Selection 2004 17(2):113-117; doi:10.1093/protein/gzh016
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© 2004 Oxford University Press
The influence of the region between residues 220 and 344 and beyond in Phrixotrix railroad worm luciferases green and red bioluminescence
1Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA, 2Department of Biology, Instituto de Biociências, Universidade Estadual de São Paulo (UNESP), Rua 24 A, PO Box 199, Rio Claro, SP, Brazil and 4Cell Dynamics Research Group, Division of Human Life Technology, National Institute of Advanced Industrial Science and Technology, Osaka, Japan
3 To whom correspondence should be addressed at: Department of Biology, Instituto de Biociências, Universidade Estadual de São Paulo (UNESP), Rua 24 A, 1515, Bela Vista, Rio Claro, SP, 13506-900, Brazil e-mail: viviani{at}rc.unesp.br
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; 
max = 548 nm) green light-emitting luciferase and Phrixothrix hirtus (PxhRE; max = 623 nm) red light-emitting luciferases. The emission spectrum (max = 571 nm) and KM 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 (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.
Received January 24, 2003; revised November 24, 2003; accepted November 28, 2003 Edited by Alan Fersht
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