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A structural basis for reversible photoswitching of absorbance spectra in red fluorescent protein rsTagRFP.
J Mol Biol 2012; 417(3):144-51JM

Abstract

rsTagRFP is the first monomeric red fluorescent protein (FP) with reversibly photoswitchable absorbance spectra. The switching is realized by irradiation of rsTagRFP with blue (440 nm) and yellow (567 nm) light, turning the protein fluorescence ON and OFF, respectively. It is perhaps the most useful probe in this color class that has yet been reported. Because of the photoswitchable absorbance, rsTagRFP can be used as an acceptor in photochromic Förster resonance energy transfer. Yellow FPs, YPet and mVenus, are demonstrated to be excellent photochromic Förster resonance energy transfer donors for the rsTagRFP acceptor in its fusion constructs. Analysis of X-ray structures has shown that photoswitching of rsTagRFP is accompanied by cis-trans isomerization and protonation/deprotonation of the chromophore, with the deprotonated cis- and protonated trans-isomers corresponding to its ON and OFF states, respectively. Unlike in other photoswitchable FPs, both conformers of rsTagRFP chromophore are essentially coplanar. Two other peculiarities of the rsTagRFP chromophore are an essentially hydrophobic environment of its p-hydroxyphenyl site and the absence of direct hydrogen bonding between this moiety and the protein scaffold. The influence of the immediate environment on rsTagRFP chromophore was probed by site-directed mutagenesis. Residues Glu145 and His197 were found to participate in protonation/deprotonation of the chromophore accompanying the photoswitching of rsTagRFP fluorescence, whereas residues Met160 and Leu174 were shown to spatially restrict chromophore isomerization, favoring its radiative decay.

Authors+Show Affiliations

Synchrotron Radiation Research Section, Macromolecular Crystallography Laboratory, National Cancer Institute, Argonne, IL 60439, USA. pletnevs@mail.nih.govNo 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, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

22310052

Citation

Pletnev, Sergei, et al. "A Structural Basis for Reversible Photoswitching of Absorbance Spectra in Red Fluorescent Protein RsTagRFP." Journal of Molecular Biology, vol. 417, no. 3, 2012, pp. 144-51.
Pletnev S, Subach FV, Dauter Z, et al. A structural basis for reversible photoswitching of absorbance spectra in red fluorescent protein rsTagRFP. J Mol Biol. 2012;417(3):144-51.
Pletnev, S., Subach, F. V., Dauter, Z., Wlodawer, A., & Verkhusha, V. V. (2012). A structural basis for reversible photoswitching of absorbance spectra in red fluorescent protein rsTagRFP. Journal of Molecular Biology, 417(3), pp. 144-51. doi:10.1016/j.jmb.2012.01.044.
Pletnev S, et al. A Structural Basis for Reversible Photoswitching of Absorbance Spectra in Red Fluorescent Protein RsTagRFP. J Mol Biol. 2012 Mar 30;417(3):144-51. PubMed PMID: 22310052.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A structural basis for reversible photoswitching of absorbance spectra in red fluorescent protein rsTagRFP. AU - Pletnev,Sergei, AU - Subach,Fedor V, AU - Dauter,Zbigniew, AU - Wlodawer,Alexander, AU - Verkhusha,Vladislav V, Y1 - 2012/01/30/ PY - 2011/10/17/received PY - 2012/01/23/revised PY - 2012/01/25/accepted PY - 2012/2/8/entrez PY - 2012/2/9/pubmed PY - 2012/5/19/medline SP - 144 EP - 51 JF - Journal of molecular biology JO - J. Mol. Biol. VL - 417 IS - 3 N2 - rsTagRFP is the first monomeric red fluorescent protein (FP) with reversibly photoswitchable absorbance spectra. The switching is realized by irradiation of rsTagRFP with blue (440 nm) and yellow (567 nm) light, turning the protein fluorescence ON and OFF, respectively. It is perhaps the most useful probe in this color class that has yet been reported. Because of the photoswitchable absorbance, rsTagRFP can be used as an acceptor in photochromic Förster resonance energy transfer. Yellow FPs, YPet and mVenus, are demonstrated to be excellent photochromic Förster resonance energy transfer donors for the rsTagRFP acceptor in its fusion constructs. Analysis of X-ray structures has shown that photoswitching of rsTagRFP is accompanied by cis-trans isomerization and protonation/deprotonation of the chromophore, with the deprotonated cis- and protonated trans-isomers corresponding to its ON and OFF states, respectively. Unlike in other photoswitchable FPs, both conformers of rsTagRFP chromophore are essentially coplanar. Two other peculiarities of the rsTagRFP chromophore are an essentially hydrophobic environment of its p-hydroxyphenyl site and the absence of direct hydrogen bonding between this moiety and the protein scaffold. The influence of the immediate environment on rsTagRFP chromophore was probed by site-directed mutagenesis. Residues Glu145 and His197 were found to participate in protonation/deprotonation of the chromophore accompanying the photoswitching of rsTagRFP fluorescence, whereas residues Met160 and Leu174 were shown to spatially restrict chromophore isomerization, favoring its radiative decay. SN - 1089-8638 UR - https://www.unboundmedicine.com/medline/citation/22310052/A_structural_basis_for_reversible_photoswitching_of_absorbance_spectra_in_red_fluorescent_protein_rsTagRFP_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(12)00098-8 DB - PRIME DP - Unbound Medicine ER -