JBC Invitrogen Ultrasensitive Cytokine Assays

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Originally published In Press as doi:10.1074/jbc.C100694200 on December 31, 2001

J. Biol. Chem., Vol. 277, Issue 10, 7633-7636, March 8, 2002
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ACCELERATED PUBLICATION
Analysis of DsRed Mutants
SPACE AROUND THE FLUOROPHORE ACCELERATES FLUORESCENCE DEVELOPMENT*

Alexey V. TerskikhDagger §, Arkady F. Fradkov||, Andrey G. Zaraisky||, Andrey V. Kajava**, and Brigitte AngresDagger Dagger

From the Dagger  Stanford University, School of Medicine, Beckman Center, Stanford, California 94305, the || Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia, the ** Center for Molecular Modeling, Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892, and the Dagger Dagger  BD Biosciences Clontech, Palo Alto, California 94303

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.

* This work was partially supported by Russian Federation of Fundamental Investigations Grant N 01-04-49037 (to A. F. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Pathology, Stanford University, 279 Campus Dr., Stanford, CA 94305. E-mail: Alexey.Terskikh@Stanford.edu.

These authors contributed equally to this work.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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