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1 August 2001 Functional Characterization of Permuted Enhanced Green Fluorescent Proteins Comprising Varying Linker Peptides
Walther Akemann, Christopher Dinesh Raj, Thomas Knöpfel
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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°C. Renaturation proceeded with a short (∼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.

Walther Akemann, Christopher Dinesh Raj, and Thomas Knöpfel "Functional Characterization of Permuted Enhanced Green Fluorescent Proteins Comprising Varying Linker Peptides," Photochemistry and Photobiology 74(2), 356-363, (1 August 2001).<0356:FCOPEG>2.0.CO;2
Received: 25 January 2001; Accepted: 1 May 2001; Published: 1 August 2001

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