UvA-DARE ( Digital Academic Repository ) Optimization of fluorescent proteins for novel quantitative multiparameter microscopy approaches

Enhanced cyan and yellow fluorescent proteins are widely used for dual color imaging and protein-protein interaction studies based on fluorescence resonance energy transfer. Use of these fluorescent proteins can be limited by their thermosensitivity, dim fluorescence, and tendency for aggregation. Here we report the results of a site-directed mutagenesis approach to improve these fluorescent proteins. We created monomeric optimized variants of ECFP and EYFP, which fold faster and more efficiently at 37 degrees C and have superior solubility and brightness. Bacteria expressing SCFP3A were 9-fold brighter than those expressing ECFP and 1.2-fold brighter than bacteria expressing Cerulean. SCFP3A has an increased quantum yield (0.56) and fluorescence lifetime. Bacteria expressing SYFP2 were 12 times brighter than those expressing EYFP(Q69K) and almost 2-fold brighter than bacteria expressing Venus. In HeLa cells, the improvements were less pronounced; nonetheless, cells expressing SCFP3A and SYFP2 were both 1.5-fold brighter than cells expressing ECFP and EYFP(Q69K), respectively. The enhancements of SCFP3A and SYFP2 are most probably due to an increased intrinsic brightness (1.7-fold and 1.3-fold for purified recombinant proteins, compared to ECFP & EYFP(Q69K), respectively) and due to enhanced protein folding and maturation. The latter enhancements most significantly contribute to the increased fluorescent yield in bacteria whereas they appear less significant for mammalian cell systems. SCFP3A and SYFP2 make a superior donor-acceptor pair for fluorescence resonance energy transfer, because of the high quantum yield and increased lifetime of SCFP3A and the high extinction coefficient of SYFP2. Furthermore, SCFP1, a CFP variant with a short fluorescence lifetime but identical spectra compared to ECFP and SCFP3A, was characterized. Using the large lifetime difference between SCFP1 and SCFP3A enabled us to perform for the first time dual-lifetime imaging of spectrally identical fluorescent species in living cells.

[1]  B. Reid,et al.  Chromophore formation in green fluorescent protein. , 1997, Biochemistry.

[2]  G J Brakenhoff,et al.  Image calibration in fluorescence microscopy , 2004, Journal of microscopy.

[3]  Squire,et al.  Multiple frequency fluorescence lifetime imaging microscopy , 2000, Journal of microscopy.

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

[5]  A. Weiss,et al.  Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module. , 1996, Protein engineering.

[6]  G. Patterson,et al.  Förster distances between green fluorescent protein pairs. , 2000, Analytical biochemistry.

[7]  R. Heim,et al.  Understanding structure-function relationships in the Aequorea victoria green fluorescent protein. , 1999, Methods in cell biology.

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

[9]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[10]  Robert M. Clegg,et al.  Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale , 1993 .

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

[12]  N. Fujihara,et al.  In Vivo Gene Transfer into Chicken Embryos via Primordial Germ Cells Using Green Fluorescent Protein as a Marker , 2000 .

[13]  S J Remington,et al.  Structural and spectral response of green fluorescent protein variants to changes in pH. , 1999, Biochemistry.

[14]  Marc Tramier,et al.  Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells. , 2002, Biophysical journal.

[15]  M. Mycek,et al.  Fluorescence lifetime imaging microscopy. , 2007, Methods in cell biology.

[16]  Richard N. Day,et al.  Fluorescent protein spectra. , 2001, Journal of cell science.

[17]  Daniel,et al.  Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp 41 from HIV , 2022 .

[18]  A Miyawaki,et al.  Dynamic and quantitative Ca2+ measurements using improved cameleons. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[19]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[20]  R. Tsien,et al.  Partitioning of Lipid-Modified Monomeric GFPs into Membrane Microdomains of Live Cells , 2002, Science.

[21]  J. Goedhart,et al.  Multiparameter Imaging for the Analysis of Intracellular Signaling , 2005, Chembiochem : a European journal of chemical biology.

[22]  G. Gilliland,et al.  Three‐Dimensional structure of schistosoma japonicum glutathione s‐transferase fused with a six‐amino acid conserved neutralizing epitope of gp41 from hiv , 1994, Protein science : a publication of the Protein Society.

[23]  E. V. van Munster,et al.  Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP‐fusion proteins and multimode FRET microscopy , 2004, Journal of microscopy.

[24]  E. V. van Munster,et al.  φFLIM: a new method to avoid aliasing in frequency‐domain fluorescence lifetime imaging microscopy , 2004, Journal of microscopy.

[25]  R. Clegg.  Fluorescence resonance energy transfer. , 2020, Current Opinion in Biotechnology.

[26]  A. Visser,et al.  Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins , 2005, Journal of Fluorescence.

[27]  Rainer Pepperkok,et al.  Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy , 1999, Current Biology.

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

[29]  G. Patterson,et al.  Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. , 1997, Biophysical journal.

[30]  R. Wachter,et al.  The crystal structure of the Y66L variant of green fluorescent protein supports a cyclization-oxidation-dehydration mechanism for chromophore maturation. , 2004, Biochemistry.

[31]  L. Stryer.  Fluorescence energy transfer as a spectroscopic ruler. , 1978, Annual review of biochemistry.

[32]  B. W. van der Meer.  Kappa-squared: from nuisance to new sense. , 2002, Journal of biotechnology.

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

[34]  Brian Herman,et al.  Fluorescence imaging spectroscopy and microscopy , 1996 .

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

[36]  H. Tønnesen,et al.  Corrected Emission Spectra and Quantum Yields for a Series of Fluorescent Compounds in the Visible Spectral Region , 2004, Journal of Fluorescence.

[37]  E. V. van Munster,et al.  Suppression of photobleaching‐induced artifacts in frequency‐domain FLIM by permutation of the recording order , 2004, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[38]  Th. Förster.  Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .

[39]  B. V. D. Meer.  Kappa-squared: from nuisance to new sense , 2002 .

[40]  F. Hartl,et al.  De novo folding of GFP fusion proteins: high efficiency in eukaryotes but not in bacteria. , 2005, Journal of molecular biology.

[41]  A Miyawaki,et al.  Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. , 2000, Nucleic acids research.

[42]  J. Dixon,et al.  Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. , 1991, Analytical biochemistry.

[43]  R. Tsien,et al.  Creating new fluorescent probes for cell biology , 2002, Nature Reviews Molecular Cell Biology.

[44]  Mark A Rizzo,et al.  An improved cyan fluorescent protein variant useful for FRET , 2004, Nature Biotechnology.

[45]  Gerco C Angenent,et al.  Analysis of MADS box protein–protein interactions in living plant cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[46]  Alessandro Esposito,et al.  Fluorescence Lifetime Imaging Microscopy , 2004, Current protocols in cell biology.

[47]  Takeharu Nagai,et al.  Shift anticipated in DNA microarray market , 2002, Nature Biotechnology.

[48]  A. Miyawaki,et al.  Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[49]  Jim Haseloff,et al.  Mutations that suppress the thermosensitivity of green fluorescent protein , 1996, Current Biology.

[50]  John A Tainer,et al.  Understanding GFP chromophore biosynthesis: controlling backbone cyclization and modifying post-translational chemistry. , 2005, Biochemistry.