Intrinsic dynamics in ECFP and Cerulean control fluorescence quantum yield.

Enhanced cyan fluorescent protein (ECFP) and its variant Cerulean are genetically encoded fluorophores widely used as donors in FRET-based cell imaging experiments. First, we have confirmed through denaturation experiments that the double-peak spectroscopic signature of these fluorescent proteins originates from the indole ring of the chromophore. Then, to explain the improvement in the fluorescence properties of Cerulean compared to those of ECFP, we have determined the high-resolution crystal structures of these two proteins at physiological pH and performed molecular dynamics simulations. In both proteins, the N-terminal half of the seventh strand exhibits two conformations. These conformations both have a complex set of van der Waals interactions with the chromophore and, as our simulations suggest, they interconvert on a nanosecond time scale. The Y145A and H148D mutations in Cerulean stabilize these interactions and allow the chromophore to be more planar, better packed, and less prone to collisional quenching, albeit only intermittently. As a consequence, the probability of nonradiative decay is significantly decreased. Our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins and provide insights that should allow the design of mutants with enhanced optical properties.

[1]  W. D. Mcelroy,et al.  Bioluminescence and Chemiluminescence: Basic Chemistry and Analytical Applications , 1981 .

[2]  R. Tsien,et al.  Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein , 2004, Nature Biotechnology.

[3]  Marc Albe,et al.  The dynamo library for molecular simulations using hybrid quantum mechanical and molecular mechanical potentials , 2000 .

[4]  M. Zimmer,et al.  Photophysics and Dihedral Freedom of the Chromophore in Yellow, Blue, and Green Fluorescent Protein , 2008, The journal of physical chemistry. B.

[5]  Walter Thiel,et al.  QM/MM methods for biomolecular systems. , 2009, Angewandte Chemie.

[6]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[7]  Victor S. Lobanov,et al.  High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery , 2001 .

[8]  D. Bourgeois,et al.  Reverse pH-dependence of chromophore protonation explains the large Stokes shift of the red fluorescent protein mKeima. , 2009, Journal of the American Chemical Society.

[9]  T. Holak,et al.  Slow exchange in the chromophore of a green fluorescent protein variant. , 2002, Journal of the American Chemical Society.

[10]  Christian Eggeling,et al.  Structural basis for reversible photoswitching in Dronpa , 2007, Proceedings of the National Academy of Sciences.

[11]  D. Piston,et al.  X-ray structure of Cerulean GFP: a tryptophan-based chromophore useful for fluorescence lifetime imaging. , 2007, Biochemistry.

[12]  Robert E Campbell,et al.  Structural basis for reversible photobleaching of a green fluorescent protein homologue , 2007, Proceedings of the National Academy of Sciences.

[13]  V. Adam,et al.  Advances in spectroscopic methods for biological crystals. 1. Fluorescence lifetime measurements , 2007 .

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

[15]  Jacqueline Ridard,et al.  Cyan fluorescent protein: molecular dynamics, simulations, and electronic absorption spectrum. , 2005, The journal of physical chemistry. B.

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

[17]  Janos Vörös,et al.  The density and refractive index of adsorbing protein layers. , 2004, Biophysical journal.

[18]  Joachim Goedhart,et al.  UvA-DARE ( Digital Academic Repository ) Optimization of fluorescent proteins for novel quantitative multiparameter microscopy approaches , 2007 .

[19]  Robert Huber,et al.  Expansion of the genetic code enables design of a novel "gold" class of green fluorescent proteins. , 2003, Journal of molecular biology.

[20]  W. L. Jorgensen,et al.  Development and Testing of the OPLS All-Atom Force Field on Conformational Energetics and Properties of Organic Liquids , 1996 .

[21]  Nathan C Shaner,et al.  Novel chromophores and buried charges control color in mFruits. , 2006, Biochemistry.

[22]  A. Miyawaki,et al.  Light-dependent regulation of structural flexibility in a photochromic fluorescent protein , 2008, Proceedings of the National Academy of Sciences.

[23]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[24]  M. Field,et al.  Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations , 2008, Proceedings of the National Academy of Sciences.

[25]  William L. Jorgensen,et al.  Improved semiempirical heats of formation through the use of bond and group equivalents , 2002, J. Comput. Chem..

[26]  Patrick S Daugherty,et al.  Evolutionary optimization of fluorescent proteins for intracellular FRET , 2005, Nature Biotechnology.

[27]  Christian Eggeling,et al.  Structure and mechanism of the reversible photoswitch of a fluorescent protein. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[28]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

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

[30]  Nathan C Shaner,et al.  A guide to choosing fluorescent proteins , 2005, Nature Methods.

[31]  Wolfgang Kabsch,et al.  Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants , 1993 .

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

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

[34]  J. Siegel,et al.  Imaging the environment of green fluorescent protein. , 2002, Biophysical journal.

[35]  M. Gilson,et al.  Prediction of pH-dependent properties of proteins. , 1994, Journal of molecular biology.

[36]  Zbigniew Dauter,et al.  A Crystallographic Study of Bright Far-Red Fluorescent Protein mKate Reveals pH-induced cis-trans Isomerization of the Chromophore* , 2008, Journal of Biological Chemistry.

[37]  Pascal Pernot,et al.  Complex fluorescence of the cyan fluorescent protein: comparisons with the H148D variant and consequences for quantitative cell imaging. , 2008, Biochemistry.