Circularly permuted variants of the green fluorescent protein

  title={Circularly permuted variants of the green fluorescent protein},
  author={Simon Topell and Jens Hennecke and Rudi Glockshuber},
  journal={FEBS Letters},
Circularly permuted monomeric red fluorescent proteins with new termini in the β‐sheet
The extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness is reported, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence.
Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP
By directed rational mutagenesis, a new variant of the Sapphire mutant of GFP with improved folding properties that turns out to be especially beneficial when expressed within organelles or as a fusion tag is produced.
Cyclic Green Fluorescent Protein Produced in Vivo Using an Artificially Split PI-PfuI Intein from Pyrococcus furiosus *
Green fluorescent protein (GFP) was cyclized with this method in vivo on milligram scales and might become a novel tool for studying the role of termini and backbone topology in various biological processes such as protein degradation and translocation in vivo as well as in vitro.
Engineering and characterization of a superfolder green fluorescent protein
A robustly folded version of GFP is generated, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides, and shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics.
Specific modification at the C-terminal lysine residue of the green fluorescent protein variant, GFPuv, expressed in Escherichia coli
The recombinant expression of G FPuv, a commonly-used mutant of GFP, in E. coli produced two distinct molecular species as judged by in-gel fluorescence SDS-PAGE, which could be separately purified by anion-exchange chromatography without any remarkable differences in the fluorescence spectra.
Functional Characterization of Permuted Enhanced Green Fluorescent Proteins Comprising Varying Linker Peptides ¶
Laser‐scanning microscopy of HeLa cells demonstrated that the PEGFP are particularly well suited as fluorescent indicators in two‐photon imaging, and expression dynamics of P EGFP were revealed to be similar to that of EGFP.
Tolerance of a Knotted Near-Infrared Fluorescent Protein to Random Circular Permutation.
Results show how the contact order of a knotted BphP can be altered without disrupting chromophore binding and fluorescence, an important step toward the creation of near-infrared biosensors with expanded chemical sensing functions for in vivo imaging.
Functional Characterization of Permuted Enhanced Green Fluorescent Proteins Comprising Varying Linker Peptides¶
Laser-scanning microscopy of HeLa cells demonstrated that the PEGFP are particularly well suited as fluorescent indicators in two-photon imaging and expression dynamics of PEG FP were revealed to be similar to that of EGFP.
Identification of Sites Within a Monomeric Red Fluorescent Protein that Tolerate Peptide Insertion and Testing of Corresponding Circular Permutations
This work has investigated the tolerance of an engineered monomeric descendent of Discosoma RFP, known as mCherry, towards peptide insertion and circular permutation and identified six genetically distinct sites localized in three different loops where a sequence of five residues could be inserted without abolishing the ability of the protein to form its intrinsic red fluorescent chromophore.
Understanding the folding of GFP using biophysical techniques
The recent literature on protein engineering studies that have improved the folding properties of GFP are reviewed and the biophysical work on the folding of G FP is discussed, beginning to reveal how this large and complex structure forms.


Crystal Structure of the Aequorea victoria Green Fluorescent Protein
The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The
The molecular structure of green fluorescent protein
The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 Å by multiwavelength anomalous dispersion phasing methods and the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.
Correct folding of circularly permuted variants of a beta alpha barrel enzyme in vivo.
It is shown that two circularly permuted versions of the gene of a single-domain beta alpha barrel enzyme can be expressed in Escherichia coli and are practically indistinguishable from the original enzyme by several structural and spectroscopic criteria.
Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein.
The characterization of the Aequorea victoria GFP chromophore is described, which is released as a hexapeptide upon digestion of the protein with papain, formed upon cyclization of the residues Ser-dehydroTyr-Gly within the polypeptide.
Wavelength mutations and posttranslational autoxidation of green fluorescent protein.
  • R. Heim, D. Prasher, R. Tsien
  • Biology, Chemistry
    Proceedings of the National Academy of Sciences of the United States of America
  • 1994
The availability of two visibly distinct colors should significantly extend the usefulness of GFP in molecular and cell biology by enabling in vivo visualization of differential gene expression and protein localization and measurement of protein association by fluorescence resonance energy transfer.
Deletions of the Aequorea victoria Green Fluorescent Protein Define the Minimal Domain Required for Fluorescence*
Using deletion analysis, the minimal domain in GFP required for fluorescence to amino acids 7–229 is defined, which starts at the middle of the first small α helix at the N terminus of GFP and ends immediately following the last β sheet.
Chromophore formation in green fluorescent protein.
The green fluorescent protein (GFP) from the jellyfish Aequorea Victoria forms an intrinsic chromophore through cyclization and oxidation of an internal tripeptide motif, which concludes that GFP Chromophore formation is an autocatalytic process.