Functional characterization of permuted enhanced green fluorescent proteins comprising varying linker peptides.
New variants of green fluorescent protein (GFP) can be engineered by circular permutation of their amino acid sequence. We characterized a series of permuted enhanced GFP (PEGFP) with new termini introduced at N144-Y145 and linkers of 1, 3, 5 and 6 residues inserted between G232 and M1, as well as a variant with an extended 7-residues linker between K238 and M1. A minimum linker length of 3 residues was necessary for a functional chromophore to be formed, and linkers exceeding 4 residues yielded almost the same fluorescence quantum yield as enhanced GFP (EGFP). PEGFP exhibited dual-wavelength absorption and fluorescence excitation with peaks at 395 and 490 nm but single-wavelength emission at 512 nm. Fluorescence emission increased with increasing pH for all excitation wavelengths with a pKa of 7.7. Between the pH values of 6 and 8 optical absorption showed an isobestic point at 445 nm. PEGFP rapidly denatured in urea between 50 and 60 degrees C. Renaturation proceeded with a short (approximately 29 s) and a longer (> 150 s) time constant. Transient transfection of HEK293 and HeLa cells revealed the expression dynamics of PEGFP to be similar to that of EGFP. Laser-scanning microscopy of HeLa cells demonstrated that the PEGFP are particularly well suited as fluorescent indicators in two-photon imaging.
Akemann
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Laboratory for Neuronal Circuit Dynamics, RIKEN Brain Science Institute (BSI), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
Raj
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Knöpfel
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D010777:Photochemistry; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D017931:DNA Primers; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins