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Semi-rational engineering of a coral fluorescent protein into an efficient highlighter.
EMBO Rep. 2005 Mar; 6(3):233-8.ER

Abstract

Kaede is a natural photoconvertible fluorescent protein found in the coral Trachyphyllia geoffroyi. It contains a tripeptide, His 62-Tyr 63-Gly 64, which acts as a green chromophore that is photoconvertible to red following (ultra-) violet irradiation. Here, we report the molecular cloning and crystal structure determination of a new fluorescent protein, KikG, from the coral Favia favus, and its in vitro evolution conferring green-to-red photoconvertibility. Substitution of the His 62-Tyr 63-Gly 64 sequence into the native protein provided only negligible photoconversion. On the basis of the crystal structure, semi-rational mutagenesis of the amino acids surrounding the chromophore was performed, leading to the generation of an efficient highlighter, KikGR. Within mammalian cells, KikGR is more efficiently photoconverted and is several-fold brighter in both the green and red states than Kaede. In addition, KikGR was successfully photoconverted using two-photon excitation microscopy at 760 nm, ensuring optical cell labelling with better spatial discrimination in thick and highly scattering tissues.

Authors+Show Affiliations

Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15731765

Citation

Tsutsui, Hidekazu, et al. "Semi-rational Engineering of a Coral Fluorescent Protein Into an Efficient Highlighter." EMBO Reports, vol. 6, no. 3, 2005, pp. 233-8.
Tsutsui H, Karasawa S, Shimizu H, et al. Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep. 2005;6(3):233-8.
Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N., & Miyawaki, A. (2005). Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Reports, 6(3), 233-8.
Tsutsui H, et al. Semi-rational Engineering of a Coral Fluorescent Protein Into an Efficient Highlighter. EMBO Rep. 2005;6(3):233-8. PubMed PMID: 15731765.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. AU - Tsutsui,Hidekazu, AU - Karasawa,Satoshi, AU - Shimizu,Hideaki, AU - Nukina,Nobuyuki, AU - Miyawaki,Atsushi, PY - 2004/10/29/received PY - 2005/01/21/revised PY - 2005/01/31/accepted PY - 2005/2/26/pubmed PY - 2005/8/9/medline PY - 2005/2/26/entrez SP - 233 EP - 8 JF - EMBO reports JO - EMBO Rep. VL - 6 IS - 3 N2 - Kaede is a natural photoconvertible fluorescent protein found in the coral Trachyphyllia geoffroyi. It contains a tripeptide, His 62-Tyr 63-Gly 64, which acts as a green chromophore that is photoconvertible to red following (ultra-) violet irradiation. Here, we report the molecular cloning and crystal structure determination of a new fluorescent protein, KikG, from the coral Favia favus, and its in vitro evolution conferring green-to-red photoconvertibility. Substitution of the His 62-Tyr 63-Gly 64 sequence into the native protein provided only negligible photoconversion. On the basis of the crystal structure, semi-rational mutagenesis of the amino acids surrounding the chromophore was performed, leading to the generation of an efficient highlighter, KikGR. Within mammalian cells, KikGR is more efficiently photoconverted and is several-fold brighter in both the green and red states than Kaede. In addition, KikGR was successfully photoconverted using two-photon excitation microscopy at 760 nm, ensuring optical cell labelling with better spatial discrimination in thick and highly scattering tissues. SN - 1469-221X UR - https://www.unboundmedicine.com/medline/citation/15731765/Semi_rational_engineering_of_a_coral_fluorescent_protein_into_an_efficient_highlighter_ L2 - https://doi.org/10.1038/sj.embor.7400361 DB - PRIME DP - Unbound Medicine ER -