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A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria)

A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria)
A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria)
We performed the biochemical and biophysical characterization of a red fluorescent protein, eqFP611, from the sea anemone Entacmaea quadricolor cloned in Escherichia coli. With an excitation maximum at 559 nm and an emission maximum at 611 nm, the recombinant protein shows the most red-shifted emission and the largest Stokes shift of all nonmodified proteins in the green fluorescent protein family. The protein fluoresces with a high quantum yield of 0.45, although it resembles the nonfluorescent members of this protein class, as inferred from the absence of the key amino acid serine at position 143. Fluorescence is constant within the range pH 4-10. Red fluorophore maturation reaches a level of 90% after 12 h by passing through a green intermediate. After complete maturation, only a small fraction of the green species (less than 1%) persists. The protein has a reduced tendency to oligomerize, as shown by its monomeric appearance in SDS/PAGE analysis and single-molecule experiments. However, it forms tetramers at higher concentrations in the absence of detergent. Fluorescence correlation spectroscopy reveals light-driven transitions between bright and dark states on submillisecond and millisecond time scales. Applicability of eqFP611 for in vivo labeling in eukaryotic systems was shown by expression in a mammalian cell culture
0027-8424
11646-11651
Wiedenmann, J
ad445af2-680f-4927-90b3-589ac9d538f7
Schenk, A
d093de24-6f6b-4cbc-abe4-dad4011691f8
Rocker, C
60097535-cb39-4631-a53b-8f2baa42026e
Girod, A
fb7c944b-87d6-4c34-aafc-69a8cf0da0a3
Spindler, K-D
a15ff8dd-c007-429e-87f0-96f60492c13a
Nienhaus, G.U.
8f2b40ea-be57-4b32-880f-8a0e5c1585aa
Wiedenmann, J
ad445af2-680f-4927-90b3-589ac9d538f7
Schenk, A
d093de24-6f6b-4cbc-abe4-dad4011691f8
Rocker, C
60097535-cb39-4631-a53b-8f2baa42026e
Girod, A
fb7c944b-87d6-4c34-aafc-69a8cf0da0a3
Spindler, K-D
a15ff8dd-c007-429e-87f0-96f60492c13a
Nienhaus, G.U.
8f2b40ea-be57-4b32-880f-8a0e5c1585aa

Wiedenmann, J, Schenk, A, Rocker, C, Girod, A, Spindler, K-D and Nienhaus, G.U. (2002) A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria). Proceedings of the National Academy of Sciences of the United States of America, 99 (18), 11646-11651. (doi:10.1073/pnas.182157199).

Record type: Article

Abstract

We performed the biochemical and biophysical characterization of a red fluorescent protein, eqFP611, from the sea anemone Entacmaea quadricolor cloned in Escherichia coli. With an excitation maximum at 559 nm and an emission maximum at 611 nm, the recombinant protein shows the most red-shifted emission and the largest Stokes shift of all nonmodified proteins in the green fluorescent protein family. The protein fluoresces with a high quantum yield of 0.45, although it resembles the nonfluorescent members of this protein class, as inferred from the absence of the key amino acid serine at position 143. Fluorescence is constant within the range pH 4-10. Red fluorophore maturation reaches a level of 90% after 12 h by passing through a green intermediate. After complete maturation, only a small fraction of the green species (less than 1%) persists. The protein has a reduced tendency to oligomerize, as shown by its monomeric appearance in SDS/PAGE analysis and single-molecule experiments. However, it forms tetramers at higher concentrations in the absence of detergent. Fluorescence correlation spectroscopy reveals light-driven transitions between bright and dark states on submillisecond and millisecond time scales. Applicability of eqFP611 for in vivo labeling in eukaryotic systems was shown by expression in a mammalian cell culture

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More information

Published date: 3 September 2002
Organisations: Ocean and Earth Science

Identifiers

Local EPrints ID: 46726
URI: http://eprints.soton.ac.uk/id/eprint/46726
ISSN: 0027-8424
PURE UUID: 97974706-6b0d-4424-aeb3-62cfe27f2691
ORCID for J Wiedenmann: ORCID iD orcid.org/0000-0003-2128-2943

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Date deposited: 16 Jul 2007
Last modified: 10 Nov 2021 03:18

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Contributors

Author: J Wiedenmann ORCID iD
Author: A Schenk
Author: C Rocker
Author: A Girod
Author: K-D Spindler
Author: G.U. Nienhaus

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