Structural Tuning of the Fluorescent Protein iLOV for Improved Photostability*
- John M. Christie‡§,1,2,
- Kenichi Hitomi‡¶‖,1,
- Andrew S. Arvai‡,
- Kimberly A. Hartfield‡,
- Marcel Mettlen**,
- Ashley J. Pratt‡,
- John A. Tainerঠand
- Elizabeth D. Getzoff‡,3
- From the ‡Department of Molecular Biology and Skaggs Institute for Chemical Biology and
- the **Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037,
- the §Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom,
- the ¶Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, and
- the ‖Section of Laboratory Equipment, National Institute of Biomedical Innovation, 7-6-8, Saito-Asagi, Ibaraki, Osaka 567-0085, Japan
- ↵2 To whom correspondence may be addressed: Institute of Molecular Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow. Tel.: 44-141-330-2392; Fax: 44-141-330-4447; E-mail: john.christie{at}glasgow.ac.uk.
- ↵3 To whom correspondence may be addressed. Tel.: 858-784-2878; Fax: 858-784-2289; E-mail: edg{at}scripps.edu.
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↵1 Both authors contributed equally to this work.
Abstract
Fluorescent proteins derived from light, oxygen, or voltage (LOV) domains offer advantages over green fluorescent protein (GFP) from their small size and efficacy under anaerobic conditions. The flavoprotein improved LOV (iLOV) was engineered from the blue light receptor phototropin as a reporter of viral infection. To inform the molecular basis for the improved, photoreversible, fluorescent properties of iLOV, we employed directed evolution and determined five LOV crystallographic structures. Comparative structural analyses between iLOV and its progenitors reveal mutation-induced constraints in the environment of the flavin mononucleotide (FMN) chromophore; in iLOV, the methyl group of Thr-394 “crowds” the FMN isoalloxazine ring, Leu-470 triggers side chain “flipping” of Leu-472, and the terminal FMN phosphate shows increased anchoring. We further engineered iLOV variants that are readily detectable in bacterial and mammalian cells due to order-of-magnitude photostability increases. Structure determination of a resulting representative photostable iLOV (phiLOV) variant reveals additional constraints on the chromophore. Aromatic residues Tyr-401 and Phe-485 in phiLOV sandwich the FMN isoalloxazine ring from both sides, whereas Ser-390 anchors the side chain of FMN-interacting Gln-489 Our combined structural and mutational results reveal that constraining the FMN fluorophore yields improved photochemical properties for iLOV and its new photostable derivative. These findings provide a framework for structural fine-tuning of LOV scaffold proteins to maximize their potential as oxygen-independent fluorescent reporters.
Footnotes
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↵* This work was supported by the award of a Royal Society University Research Fellowship (to J. M. C.), National Institutes of Health Grant GM37684 (to E. D. G.), and a grant from the Skaggs Institute for Chemical Biology (to K. H.).
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This article contains supplemental Tables S1–S3 and Figs. S1–S8.
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The atomic coordinates and structure factors (codes 4EEP, 4EER, 4EES, 4EET, and 4EEU) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
- Received November 1, 2011.
- Revision received March 17, 2012.
- © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
