Optimized and far-red-emitting variants of fluorescent protein eqFP611.
Fluorescent proteins (FPs) emitting in the far-red region of the spectrum are highly advantageous for whole-body imaging applications because scattering and absorption of long-wavelength light is markedly reduced in tissue. We characterized variants of the red fluorescent protein eqFP611 with bright fluorescence emission shifted up to 639 nm. The additional red shift is caused by a trans-cis isomerization of the chromophore. The equilibrium between the trans and cis conformations is strongly influenced by amino acid residues 143 and 158. Pseudo monomeric tags were obtained by further genetic engineering. For the red chromophores of eqFP611 variants, molar extinction coefficients of up to approximately 150,000 were determined by an approach that is not affected by the presence of molecules with nonfunctional red chromophores. The bright fluorescence makes the red-shifted eqFP611 variants promising lead structures for the development of near-infrared fluorescent markers. The red fluorescent proteins performed well in cell biological applications, including two-photon imaging.
Kredel
Simone
S
Institute of General Zoology and Endocrinology, University of Ulm, 89069 Ulm, Germany.
Nienhaus
Karin
K
Oswald
Franz
F
Wolff
Michael
M
Ivanchenko
Sergey
S
Cymer
Florian
F
Jeromin
Andreas
A
Michel
Francois J
FJ
Spindler
Klaus-Dieter
KD
Heilker
Ralf
R
Nienhaus
G Ulrich
GU
Wiedenmann
Jörg
J
D000042:Absorption; D015415:Biomarkers / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D020836:Protein Structure, Quaternary; D013997:Time Factors; D000097573:Red Fluorescent Protein
D015415:Biomarkers; D008164:Luminescent Proteins; D050505:Mutant Proteins; D000097573:Red Fluorescent Protein