Analysis of DsRed Mutants. Space around the fluorophore accelerates fluorescence development.
Earlier mutagenesis of the red fluorescent protein drFP583, also called DsRed, resulted in a mutant named Fluorescent Timer (Terskikh, A., Fradkov, A., Ermakova, G., Zaraisky, A., Tan, P., Kajava, A. V., Zhao, X., Lukyanov, S., Matz, M., Kim, S., Weissman, I., and Siebert, P. (2000) Science 290, 1585--1588). Further mutagenesis generated variants with novel and improved fluorescent properties. The mutant called AG4 exhibits only green fluorescence. The mutant, called E5up (V105A), shows complete fluorophore maturation, eventually eliminating residual green fluorescence present in DsRed. Finally, the mutant, called E57 (V105A, I161T, S197A), matures faster than DsRed as demonstrated in vitro with purified protein and in vivo with recombinant protein expressed in Escherichia coli and Xenopus leavis. Comparative analysis of the mutants in the context of the crystal structure of DsRed suggests that mutants with free space around the fluorophore mature faster and more completely.
Terskikh
Alexey V
AV
Stanford University, School of Medicine, Beckman Center, Stanford, California 94305, USA. Alexey.Terskikh@Stanford.edu
Fradkov
Arkady F
AF
Zaraisky
Andrey G
AG
Kajava
Andrey V
AV
Angres
Brigitte
B
D000818:Animals; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D049452:Green Fluorescent Proteins; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors; D014982:Xenopus laevis
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins