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

被引:199
作者
Wiedenmann, J
Schenk, A
Röcker, C
Girod, A
Spindler, KD
Nienhaus, GU [1 ]
机构
[1] Univ Ulm, Dept Biophys, D-89069 Ulm, Germany
[2] Univ Ulm, Dept Gen Zool & Endocrinol, D-89069 Ulm, Germany
[3] European Mol Biol Lab, Cell Biol & Cell Biophys Programme, D-69126 Heidelberg, Germany
[4] Univ Illinois, Dept Phys, Urbana, IL 61801 USA
关键词
D O I
10.1073/pnas.182157199
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 approximate to12 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.
引用
收藏
页码:11646 / 11651
页数:6
相关论文
共 29 条
[1]   Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral [J].
Baird, GS ;
Zacharias, DA ;
Tsien, RY .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (22) :11984-11989
[2]   Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed) [J].
Bevis, BJ ;
Glick, BS .
NATURE BIOTECHNOLOGY, 2002, 20 (01) :83-87
[3]   A monomeric red fluorescent protein [J].
Campbell, RE ;
Tour, O ;
Palmer, AE ;
Steinbach, PA ;
Baird, GS ;
Zacharias, DA ;
Tsien, RY .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2002, 99 (12) :7877-7882
[4]   GREEN FLUORESCENT PROTEIN AS A MARKER FOR GENE-EXPRESSION [J].
CHALFIE, M ;
TU, Y ;
EUSKIRCHEN, G ;
WARD, WW ;
PRASHER, DC .
SCIENCE, 1994, 263 (5148) :802-805
[5]   Identification of different emitting species in the red fluorescent protein DsRed by means of ensemble and single-molecule spectroscopy [J].
Cotlet, M ;
Hofkens, J ;
Habuchi, S ;
Dirix, G ;
Van Guyse, M ;
Michiels, J ;
Vanderleyden, J ;
De Schryver, FC .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2001, 98 (25) :14398-14403
[6]   Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence [J].
Fradkov, AF ;
Chen, Y ;
Ding, L ;
Barsova, EV ;
Matz, MV ;
Lukyanov, SA .
FEBS LETTERS, 2000, 479 (03) :127-130
[7]   GFP-like chromoproteins as a source of far-red fluorescent proteins [J].
Gurskaya, NG ;
Fradkov, AF ;
Terskikh, A ;
Matz, MV ;
Labas, YA ;
Martynov, VI ;
Yanushevich, YG ;
Lukyanov, KA ;
Lukyanov, SA .
FEBS LETTERS, 2001, 507 (01) :16-20
[8]   Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy [J].
Haupts, U ;
Maiti, S ;
Schwille, P ;
Webb, WW .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1998, 95 (23) :13573-13578
[9]   Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: Coral red (dsRed) and yellow (Citrine) [J].
Heikal, AA ;
Hess, ST ;
Baird, GS ;
Tsien, RY ;
Webb, WW .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2000, 97 (22) :11996-12001
[10]   Photophysics of DsRed, a red fluorescent protein, from the ensemble to the single-molecule level [J].
Lounis, B ;
Deich, J ;
Rosell, FI ;
Boxer, SG ;
Moerner, WE .
JOURNAL OF PHYSICAL CHEMISTRY B, 2001, 105 (21) :5048-5054