Structure and mechanism of the reversible photoswitch of a fluorescent protein.

Proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state bear enormous potential in diverse fields, such as data storage, in vivo protein tracking, and subdiffraction resolution light microscopy. However, these proteins could hitherto not live up to their full potential because the molecular switching mechanism is not resolved. Here, we clarify the molecular photoswitching mechanism of asFP595, a green fluorescent protein (GFP)-like protein that can be transferred from a nonfluorescent "off" to a fluorescent "on" state and back again, by green and blue light, respectively. To this end, we establish reversible photoswitching of fluorescence in whole protein crystals and show that the switching kinetics in the crystal is identical with that in solution. Subsequent x-ray analysis demonstrated that upon the absorption of a green photon, the chromophore isomerizes from a trans (off) to a cis (on) state. Molecular dynamics calculations suggest that isomerization occurs through a bottom hula twist mechanism with concomitant rotation of both bonds of the chromophoric methine ring bridge. This insight into the switching mechanism should facilitate the targeted design of photoswitchable proteins. Reversible photoswitching of the protein chromophore system within intact crystals also constitutes a step toward the use of fluorescent proteins in three-dimensional data recording.

[1]  Berk Hess,et al.  GROMACS 3.0: a package for molecular simulation and trajectory analysis , 2001 .

[2]  P. Tavan,et al.  Ligand Binding: Molecular Mechanics Calculation of the Streptavidin-Biotin Rupture Force , 1996, Science.

[3]  Massimo Olivucci,et al.  Origin, nature, and fate of the fluorescent state of the green fluorescent protein chromophore at the CASPT2//CASSCF resolution. , 2004, Journal of the American Chemical Society.

[4]  Robert S. H. Liu,et al.  A bioorganic view of the chemistry of vision: H.T.-n and B.P.-m,n mechanisms for reactions of confined, anchored polyenes , 1986 .

[5]  Marcus Dyba,et al.  Concepts for nanoscale resolution in fluorescence microscopy , 2004, Current Opinion in Neurobiology.

[6]  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.

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

[8]  J. Rossjohn,et al.  Variations on the GFP Chromophore , 2005, Journal of Biological Chemistry.

[9]  S. Boxer,et al.  Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[10]  A. Miyawaki,et al.  Regulated Fast Nucleocytoplasmic Shuttling Observed by Reversible Protein Highlighting , 2004, Science.

[11]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[12]  T. Darden,et al.  Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems , 1993 .

[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]  Andrey Yu Gorokhovatsky,et al.  Traditional GFP-type cyclization and unexpected fragmentation site in a purple chromoprotein from Anemonia sulcata, asFP595. , 2004, Biochemistry.

[15]  S. Hell,et al.  Imaging and writing at the nanoscale with focused visible light through saturable optical transitions , 2003 .

[16]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[17]  W. L. Jorgensen,et al.  The OPLS [optimized potentials for liquid simulations] potential functions for proteins, energy minimizations for crystals of cyclic peptides and crambin. , 1988, Journal of the American Chemical Society.

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

[19]  S. Lukyanov,et al.  Chromophore Environment Provides Clue to “Kindling Fluorescent Protein” Riddle* , 2003, The Journal of Biological Chemistry.

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

[21]  S. Hell.  Toward fluorescence nanoscopy , 2003, Nature Biotechnology.

[22]  G. Phillips,et al.  The molecular structure of green fluorescent protein , 1996, Nature Biotechnology.

[23]  R. Ranganathan,et al.  The structural basis for red fluorescence in the tetrameric GFP homolog DsRed , 2000, Nature Structural Biology.

[24]  Mark Prescott,et al.  The 2.0-Å Crystal Structure of eqFP611, a Far Red Fluorescent Protein from the Sea Anemone Entacmaea quadricolor* , 2003, Journal of Biological Chemistry.