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Targeted green-red photoconversion of EosFP, a fluorescent marker protein

Targeted green-red photoconversion of EosFP, a fluorescent marker protein
Targeted green-red photoconversion of EosFP, a fluorescent marker protein
EosFP is a novel fluorescent protein from the stony coral Lobophyllia hemprichii. Its gene was cloned in Escherichia coli to express the tetrameric wild-type protein. The protein emits strong green fluorescence (516 nm) that shifts toward red (581 nm) upon near-ultraviolet irradiation at ?390 nm due to a photo-induced modification that involves a break in the peptide backbone next to the chromophore. Using site-directed mutagenesis, dimeric (d1EosFP, d2EosFP) and monomeric (mEosFP) variants were produced with essentially unaltered spectroscopic properties. Here we present a spectroscopic characterization of EosFP and its variants, including room- and low-temperature spectra, fluorescence lifetime determinations, two-photon excitation and two-photon photoconversion. Furthermore, by transfection of a human cancer (HeLa) cell with a fusion construct of a mitochondrial targeting sequence and d2EosFP, we demonstrate how localized photoconversion of EosFP can be employed for resolving intracellular processes.
fluorescent protein - fusion protein - two-photon excitation - fluorescence spectroscopy - photoconversion
0092-0606
249-259
Ivanchenko, S.
99165146-f345-45ef-9f34-e72e8af01c41
Röcker, C.
1a2dcd7a-3e54-4663-a32a-50efff9e118d
Oswald, F.
8fea64d4-21b7-4f41-93dd-0c4ce331c84b
Wiedenmann, J.
ad445af2-680f-4927-90b3-589ac9d538f7
Nienhaus, G.U.
8f2b40ea-be57-4b32-880f-8a0e5c1585aa
Ivanchenko, S.
99165146-f345-45ef-9f34-e72e8af01c41
Röcker, C.
1a2dcd7a-3e54-4663-a32a-50efff9e118d
Oswald, F.
8fea64d4-21b7-4f41-93dd-0c4ce331c84b
Wiedenmann, J.
ad445af2-680f-4927-90b3-589ac9d538f7
Nienhaus, G.U.
8f2b40ea-be57-4b32-880f-8a0e5c1585aa

Ivanchenko, S., Röcker, C., Oswald, F., Wiedenmann, J. and Nienhaus, G.U. (2005) Targeted green-red photoconversion of EosFP, a fluorescent marker protein. Journal of Biological Physics, 31 (3-4), 249-259. (doi:10.1007/s10867-005-0174-z).

Record type: Article

Abstract

EosFP is a novel fluorescent protein from the stony coral Lobophyllia hemprichii. Its gene was cloned in Escherichia coli to express the tetrameric wild-type protein. The protein emits strong green fluorescence (516 nm) that shifts toward red (581 nm) upon near-ultraviolet irradiation at ?390 nm due to a photo-induced modification that involves a break in the peptide backbone next to the chromophore. Using site-directed mutagenesis, dimeric (d1EosFP, d2EosFP) and monomeric (mEosFP) variants were produced with essentially unaltered spectroscopic properties. Here we present a spectroscopic characterization of EosFP and its variants, including room- and low-temperature spectra, fluorescence lifetime determinations, two-photon excitation and two-photon photoconversion. Furthermore, by transfection of a human cancer (HeLa) cell with a fusion construct of a mitochondrial targeting sequence and d2EosFP, we demonstrate how localized photoconversion of EosFP can be employed for resolving intracellular processes.

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More information

Published date: December 2005
Keywords: fluorescent protein - fusion protein - two-photon excitation - fluorescence spectroscopy - photoconversion

Identifiers

Local EPrints ID: 51186
URI: https://eprints.soton.ac.uk/id/eprint/51186
ISSN: 0092-0606
PURE UUID: b28bea03-4c3e-4cac-93f0-e20a7e04ad4b
ORCID for J. Wiedenmann: ORCID iD orcid.org/0000-0003-2128-2943

Catalogue record

Date deposited: 08 May 2008
Last modified: 14 Mar 2019 01:40

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