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A crystallographic study of bright far-red fluorescent protein mKate reveals pH-induced cis-trans isomerization of the chromophore.
J Biol Chem 2008; 283(43):28980-7JB

Abstract

The far-red fluorescent protein mKate (lambda(ex), 588 nm; lambda(em), 635 nm; chromophore-forming triad Met(63)-Tyr(64)-Gly(65)), originating from wild-type red fluorescent progenitor eqFP578 (sea anemone Entacmaea quadricolor), is monomeric and characterized by the pronounced pH dependence of fluorescence, relatively high brightness, and high photostability. The protein has been crystallized at a pH ranging from 2 to 9 in three space groups, and four structures have been determined by x-ray crystallography at the resolution of 1.75-2.6 A. The pH-dependent fluorescence of mKate has been shown to be due to reversible cis-trans isomerization of the chromophore phenolic ring. In the non-fluorescent state at pH 2.0, the chromophore of mKate is in the trans-isomeric form. The weakly fluorescent state of the protein at pH 4.2 is characterized by a mixture of trans and cis isomers. The chromophore in a highly fluorescent state at pH 7.0/9.0 adopts the cis form. Three key residues, Ser(143), Leu(174), and Arg(197) residing in the vicinity of the chromophore, have been identified as being primarily responsible for the far-red shift in the spectra. A group of residues consisting of Val(93), Arg(122), Glu(155), Arg(157), Asp(159), His(169), Ile(171), Asn(173), Val(192), Tyr(194), and Val(216), are most likely responsible for the observed monomeric state of the protein in solution.

Authors+Show Affiliations

Macromolecular Crystallography Laboratory, NCI, National Institutes of Health, Argonne, Illinois 60439, USA. svp@ncifcrf.govNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

18682399

Citation

Pletnev, Sergei, et al. "A Crystallographic Study of Bright Far-red Fluorescent Protein mKate Reveals pH-induced Cis-trans Isomerization of the Chromophore." The Journal of Biological Chemistry, vol. 283, no. 43, 2008, pp. 28980-7.
Pletnev S, Shcherbo D, Chudakov DM, et al. A crystallographic study of bright far-red fluorescent protein mKate reveals pH-induced cis-trans isomerization of the chromophore. J Biol Chem. 2008;283(43):28980-7.
Pletnev, S., Shcherbo, D., Chudakov, D. M., Pletneva, N., Merzlyak, E. M., Wlodawer, A., ... Pletnev, V. (2008). A crystallographic study of bright far-red fluorescent protein mKate reveals pH-induced cis-trans isomerization of the chromophore. The Journal of Biological Chemistry, 283(43), pp. 28980-7. doi:10.1074/jbc.M800599200.
Pletnev S, et al. A Crystallographic Study of Bright Far-red Fluorescent Protein mKate Reveals pH-induced Cis-trans Isomerization of the Chromophore. J Biol Chem. 2008 Oct 24;283(43):28980-7. PubMed PMID: 18682399.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A crystallographic study of bright far-red fluorescent protein mKate reveals pH-induced cis-trans isomerization of the chromophore. AU - Pletnev,Sergei, AU - Shcherbo,Dmitry, AU - Chudakov,Dmitry M, AU - Pletneva,Nadezhda, AU - Merzlyak,Ekaterina M, AU - Wlodawer,Alexander, AU - Dauter,Zbigniew, AU - Pletnev,Vladimir, Y1 - 2008/08/04/ PY - 2008/8/7/pubmed PY - 2008/12/17/medline PY - 2008/8/7/entrez SP - 28980 EP - 7 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 283 IS - 43 N2 - The far-red fluorescent protein mKate (lambda(ex), 588 nm; lambda(em), 635 nm; chromophore-forming triad Met(63)-Tyr(64)-Gly(65)), originating from wild-type red fluorescent progenitor eqFP578 (sea anemone Entacmaea quadricolor), is monomeric and characterized by the pronounced pH dependence of fluorescence, relatively high brightness, and high photostability. The protein has been crystallized at a pH ranging from 2 to 9 in three space groups, and four structures have been determined by x-ray crystallography at the resolution of 1.75-2.6 A. The pH-dependent fluorescence of mKate has been shown to be due to reversible cis-trans isomerization of the chromophore phenolic ring. In the non-fluorescent state at pH 2.0, the chromophore of mKate is in the trans-isomeric form. The weakly fluorescent state of the protein at pH 4.2 is characterized by a mixture of trans and cis isomers. The chromophore in a highly fluorescent state at pH 7.0/9.0 adopts the cis form. Three key residues, Ser(143), Leu(174), and Arg(197) residing in the vicinity of the chromophore, have been identified as being primarily responsible for the far-red shift in the spectra. A group of residues consisting of Val(93), Arg(122), Glu(155), Arg(157), Asp(159), His(169), Ile(171), Asn(173), Val(192), Tyr(194), and Val(216), are most likely responsible for the observed monomeric state of the protein in solution. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/18682399/A_crystallographic_study_of_bright_far_red_fluorescent_protein_mKate_reveals_pH_induced_cis_trans_isomerization_of_the_chromophore_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=18682399 DB - PRIME DP - Unbound Medicine ER -