Functional characterization of permuted enhanced green fluorescent proteins comprising varying linker peptides

Photochem Photobiol. 2001 Aug;74(2):356-63. doi: 10.1562/0031-8655(2001)074<0356:fcopeg>;2.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cell Line
  • DNA Primers / genetics
  • Green Fluorescent Proteins
  • Humans
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics*
  • Molecular Sequence Data
  • Photochemistry
  • Protein Engineering
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Spectrometry, Fluorescence
  • Spectrophotometry


  • DNA Primers
  • Luminescent Proteins
  • Recombinant Proteins
  • Green Fluorescent Proteins