Comparative studies on the structure and stability of fluorescent proteins EGFP, zFP506, mRFP1, "dimer2", and DsRed1.

To obtain more information about the structural properties and conformational stabilities of GFP-like fluorescent proteins, we have undertaken a systematic analysis of series of green and red fluorescent proteins with different association states. The list of studied proteins includes EGFP (green monomer), zFP506 (green tetramer), mRFP1 (red monomer), "dimer2" (red dimer), and DsRed1 (red tetramer). Fluorescent and absorbance parameters, near-UV and visible CD spectra, the accessibility of the chromophores and tryptophans to acrylamide quenching, and the resistance of these proteins to the guanidine hydrochloride unfolding and kinetics of the approaching of the unfolding equilibrium have been compared. Tetrameric zFP506 was shown to be dramatically more stable than the EGFP monomer, assuming that association might contribute to the protein conformational stability. This assumption is most likely valid even though the sequences OF GFP and zPF506 are only approximately 25% identical. Interestingly, red FPs possessed comparable conformational stabilities, where monomeric mRFP1 was the most stable species under the equilibrium conditions, whereas the tetrameric DsRed1 possessed the slowest unfolding kinetics. Furthermore, EGFP is shown to be considerably less stable than mRFP1, whereas tetrameric zFP506 is the most stable species analyzed in this study. This means that the quaternary structure, being an important stabilizing factor, does not represent the only circumstance dictating the dramatic variations between fluorescent proteins in their conformational stabilities.

[1]  S. Lukyanov,et al.  GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. , 2004, Molecular biology and evolution.

[2]  O. V. Stepanenko,et al.  High stability of Discosoma DsRed as compared to Aequorea EGFP. , 2003, Biochemistry.

[3]  A. Visser,et al.  Circular dichroism spectroscopy of fluorescent proteins , 2002, FEBS letters.

[4]  R. Tsien,et al.  A monomeric red fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[5]  K. Lukyanov,et al.  Diversity and evolution of the green fluorescent protein family , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Konstantin A Lukyanov,et al.  A strategy for the generation of non‐aggregating mutants of Anthozoa fluorescent proteins , 2002, FEBS letters.

[7]  S. Lukyanov,et al.  GFP‐like chromoproteins as a source of far‐red fluorescent proteins , 2001, FEBS letters.

[8]  Kei Ito,et al.  An Enhanced Mutant of Red Fluorescent Protein DsRed for Double Labeling and Developmental Timer of Neural Fiber Bundle Formation* , 2001, The Journal of Biological Chemistry.

[9]  Konstantin A Lukyanov,et al.  Color transitions in coral's fluorescent proteins by site-directed mutagenesis , 2001, BMC Biochemistry.

[10]  M. Falk,et al.  Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed‐tagged proteins1 , 2001, FEBS letters.

[11]  F. Wouters,et al.  Imaging biochemistry inside cells. , 2001, Trends in cell biology.

[12]  A Miyawaki,et al.  Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer. , 2001, Biochemistry.

[13]  S J Remington,et al.  Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[14]  V. Verkhusha,et al.  Denaturation and partial renaturation of a tightly tetramerized DsRed protein under mildly acidic conditions , 2000, FEBS letters.

[15]  K. Spindler,et al.  Cracks in the beta-can: fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria). , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Irving L. Weissman,et al.  "Fluorescent timer": protein that changes color with time. , 2000, Science.

[17]  R Y Tsien,et al.  Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[18]  K. Kuwajima,et al.  Folding of green fluorescent protein and the cycle3 mutant. , 2000, Biochemistry.

[19]  S. Lukyanov,et al.  Natural Animal Coloration Can Be Determined by a Nonfluorescent Green Fluorescent Protein Homolog* , 2000, The Journal of Biological Chemistry.

[20]  Y Chen,et al.  Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far‐red fluorescence , 2000, FEBS letters.

[21]  S. Lukyanov,et al.  Fluorescent proteins from nonbioluminescent Anthozoa species , 1999, Nature Biotechnology.

[22]  S J Remington,et al.  Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein. , 1998, Structure.

[23]  Roger Y. Tsien,et al.  Crystal Structure of the Aequorea victoria Green Fluorescent Protein , 1996, Science.

[24]  Brian Seed,et al.  Codon usage limitation in the expression of HIV-1 envelope glycoprotein , 1996, Current Biology.

[25]  R. Tsien,et al.  Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer , 1996, Current Biology.

[26]  R Y Tsien,et al.  Understanding, improving and using green fluorescent proteins. , 1995, Trends in biochemical sciences.

[27]  M. Chalfie.  GREEN FLUORESCENT PROTEIN , 1995, Photochemistry and photobiology.

[28]  Roger Y. Tsien,et al.  Improved green fluorescence , 1995, Nature.

[29]  Douglas C. Youvan,et al.  Red-Shifted Excitation Mutants of the Green Fluorescent Protein , 1995, Bio/Technology.

[30]  R Y Tsien,et al.  Wavelength mutations and posttranslational autoxidation of green fluorescent protein. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[31]  F. Tsuji,et al.  Aequorea green fluorescent protein , 1994, FEBS letters.

[32]  M. Chalfie,et al.  Green fluorescent protein as a marker for gene expression. , 1994, Science.

[33]  M. J. Cormier,et al.  Primary structure of the Aequorea victoria green-fluorescent protein. , 1992, Gene.

[34]  W. Ward,et al.  Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein. , 1982, Biochemistry.

[35]  William W. Ward,et al.  SPECTRAL PERTURBATIONS OF THE AEQUOREA GREEN‐FLUORESCENT PROTEIN , 1982 .

[36]  W. Ward,et al.  Renaturation of Aequorea green-fluorescent protein , 1981 .

[37]  D. Marquardt.  An Algorithm for Least-Squares Estimation of Nonlinear Parameters , 1963 .

[38]  B. Glick,et al.  Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed) , 2002, Nature Biotechnology.

[39]  I. Kuznetsova,et al.  [A complex of apparatus and programs for the measurement of spectral, polarization and kinetic characteristics of fluorescence in solution]. , 1998, Tsitologiia.

[40]  S Falkow,et al.  FACS-optimized mutants of the green fluorescent protein (GFP). , 1996, Gene.

[41]  T. Stearns,et al.  The green revolution , 1995 .

[42]  C. Pace.  Determination and analysis of urea and guanidine hydrochloride denaturation curves. , 1986, Methods in enzymology.

[43]  Michael Zuker,et al.  Delta function convolution method (DFCM) for fluorescence decay experiments , 1985 .

[44]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[45]  W. Ward,et al.  Renaturation of Aequorea gree-fluorescent protein. , 1981, Biochemical and biophysical research communications.

[46]  C. Tanford.  Protein denaturation. , 1968, Advances in protein chemistry.

[47]  Shreedhar Gadge,et al.  THE MOLECULAR STRUCTURE OF GREEN FLUORESCENT PROTEIN , 2022 .