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 approximately 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.
Department of General Zoology, University of Ulm, 89069 Ulm, Germany.
Jörg
J
Wiedenmann
Andreas
A
Schenk
Carlheinz
C
Röcker
Andreas
A
Girod
Klaus-Dieter
KD
Spindler
G Ulrich
GU
Nienhaus
D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers; D003063:Cnidaria / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D000097573:Red Fluorescent Protein
D001704:Biopolymers; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein