Ultrafast and Low Barrier Motions in the Photoreactions of the Green Fluorescent Protein

Abstract

Green fluorescent protein (GFP) fluoresces efficiently under blue excitation despite major electrostatic rearrangements resulting from photoionization of the chromophore and neutralization of Glu-222. A competing phototransformation process, which ionizes the chromophore and decarboxylates Glu-222, mimics the electrostatic and structural changes in the fluorescence photocycle. Structural and spectroscopic analysis of the cryogenically stabilized photoproduct at 100 K and a structurally annealed intermediate of the phototransformed protein at 170 K reveals distinct structural relaxations involving protein, chromophore, solvent, and photogenerated CO2. Strong structural changes of the 100 K photoproduct after decarboxylation appear exclusively within 15 Å of the chromophore and include the electrostatically driven perturbations of Gln-69, Cys-70, and water molecules in an H-bonding network connecting the chromophore. X-ray crystallography to 1.85 Å resolution and static and picosecond time-resolved IR spectroscopy identify structural mechanisms common to phototransformation and to the fluorescence photocycle. In particular, the appearance of a 1697 cm-1 (+) difference band in both photocycle and phototransformation intermediates is a spectroscopic signature for the structural perturbation of Gln-69. This is taken as evidence for an electrostatically driven dynamic response that is common to both photoreaction pathways. The interactions between the chromophore and the perturbed residues and solvent are decreased or removed in the T203H single and T203H/Q69L double mutants, resulting in a strong reduction of the fluorescence quantum yield. This suggests that the electrostatic response to the transient formation of a buried charge in the wild type is important for the bright fluorescence. Green fluorescent protein (GFP) fluoresces efficiently under blue excitation despite major electrostatic rearrangements resulting from photoionization of the chromophore and neutralization of Glu-222. A competing phototransformation process, which ionizes the chromophore and decarboxylates Glu-222, mimics the electrostatic and structural changes in the fluorescence photocycle. Structural and spectroscopic analysis of the cryogenically stabilized photoproduct at 100 K and a structurally annealed intermediate of the phototransformed protein at 170 K reveals distinct structural relaxations involving protein, chromophore, solvent, and photogenerated CO2. Strong structural changes of the 100 K photoproduct after decarboxylation appear exclusively within 15 Å of the chromophore and include the electrostatically driven perturbations of Gln-69, Cys-70, and water molecules in an H-bonding network connecting the chromophore. X-ray crystallography to 1.85 Å resolution and static and picosecond time-resolved IR spectroscopy identify structural mechanisms common to phototransformation and to the fluorescence photocycle. In particular, the appearance of a 1697 cm-1 (+) difference band in both photocycle and phototransformation intermediates is a spectroscopic signature for the structural perturbation of Gln-69. This is taken as evidence for an electrostatically driven dynamic response that is common to both photoreaction pathways. The interactions between the chromophore and the perturbed residues and solvent are decreased or removed in the T203H single and T203H/Q69L double mutants, resulting in a strong reduction of the fluorescence quantum yield. This suggests that the electrostatic response to the transient formation of a buried charge in the wild type is important for the bright fluorescence. Green fluorescent protein (GFP) 2The abbreviations used are: GFP, green fluorescent protein; ESPT, excited state proton transfer; GFPL, lumi photoproduct state of GFP; GFPM, metastable intermediate of GFP; GFPR, relaxed photoproduct of GFP. (1Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4982) Google Scholar, 2Lippincott-Schwartz J. Snapp E. Kenworthy A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 444-456Crossref PubMed Scopus (986) Google Scholar) from Aequorea victoria is highly fluorescent with 400 nm excitation, despite major electrostatic rearrangements resulting from rapid charge transfer to the excited chromophore (3Chattoraj M. King B.A. Bublitz G.U. Boxer S.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8362-8367Crossref PubMed Scopus (796) Google Scholar). Rapid excited state proton transfer (ESPT) (4Forster T. Zeitschrift für Elektrochemie und Angewandte Physikalische Chemie. 1950; 54: 531-535Google Scholar, 5Weller A. Z. Elektrochem. 1952; 56: 662-668Google Scholar, 6Agmon N. J. Phys. Chem. A. 2005; 109: 13-35Crossref PubMed Scopus (501) Google Scholar) follows excitation of the neutral, phenolic chromophore, and the resulting excited phenolate state I* exhibits high quantum efficiency red-shifted emission at 508 nm, with a 3.0-ns lifetime (3Chattoraj M. King B.A. Bublitz G.U. Boxer S.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8362-8367Crossref PubMed Scopus (796) Google Scholar). The mechanism by which the protein environment suppresses non-radiative processes remains unexplained. Proposals for the ESPT pathway include a hydrogen bonding network connecting the chromophore phenolic oxygen to Glu-222 via a water molecule and Ser-205 (7Brejc K. Sixma T.K. Kitts P.A. Kain S.R. Tsien R.Y. Ormo M. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2306-2311Crossref PubMed Scopus (654) Google Scholar, 8Palm G.J. Zdanov A. Gaitanaris G.A. Stauber R. Pavlakis G.N. Wlodawer A. Nat. Struct. Biol. 1997; 4: 361-365Crossref PubMed Scopus (235) Google Scholar, 9Stoner-Ma D. Jaye A.A. Matousek P. Towrie M. Meech S.R. Tonge P.J. J. Am. Chem. Soc. 2005; 127: 2864-2865Crossref PubMed Scopus (170) Google Scholar). Recently, the possibility of a proton transfer pathway including Glu-222, Asp-82, and Glu-5 was put forward (10Agmon N. Biophys. J. 2005; 88: 2452-2461Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The transient infrared absorption reportedly developing at 1706 cm-1 with excitation of GFP in D2O (9Stoner-Ma D. Jaye A.A. Matousek P. Towrie M. Meech S.R. Tonge P.J. J. Am. Chem. Soc. 2005; 127: 2864-2865Crossref PubMed Scopus (170) Google Scholar) could not distinguish between these proposals. However, experimental evidence for the identity of the proton acceptor has recently been provided from comparison with the E222D mutant (11van Thor J.J. Zanetti G. Ronayne K. Towrie M. J. Phys. Chem. B. 2005; 109: 16099-16108Crossref PubMed Scopus (62) Google Scholar). Low quantum yield electron transfer from Glu-222 to the photoexcited chromophore triggers decarboxylation of the buried carboxyl group (12van Thor J.J. Gensch T. Hellingwerf K.J. Johnson L.N. Nat. Struct. Biol. 2002; 9: 37-41Crossref PubMed Scopus (202) Google Scholar). This irreversible phototransformation process competes with ESPT that initiates the dominant fluorescence photocycle. The buried carboxylate of Glu-222 stabilizes the protonated chromophore via electrostatic repulsion (13Yang F. Moss L.G. Phillips Jr., G.N. Nat. Biotechnol. 1996; 14: 1246-1251Crossref PubMed Scopus (1301) Google Scholar). Neutralization of Glu-222 by light-induced decarboxylation therefore leads to chromophore ionization in equilibrium at pH 8.0 (12van Thor J.J. Gensch T. Hellingwerf K.J. Johnson L.N. Nat. Struct. Biol. 2002; 9: 37-41Crossref PubMed Scopus (202) Google Scholar). Fig. 1 shows a simplified reaction scheme depicting the major charge redistributions for both the high quantum yield, reversible fluorescence photocycle, and the low quantum yield, irreversible, phototransformation pathway. The protein must relax to accommodate the electrostatic stress generated by chromophore ionization and neutralization of Glu-222, either in the phototransformed protein or following the ESPT reaction in the fluorescence photocycle. In the phototransformation pathway, we refer to the initial “dark” structure with a neutral chromophore (λmax = 400 nm) as GFPA, the initial “lumi” state produced by illumination at 100 K (λmax = 497 nm) as GFPL, the “meta” intermediate generated by thermal annealing of GFPL as GFPM, and the final, fully relaxed photoproduct previously characterized at 300 K (12van Thor J.J. Gensch T. Hellingwerf K.J. Johnson L.N. Nat. Struct. Biol. 2002; 9: 37-41Crossref PubMed Scopus (202) Google Scholar) as GFPR. We describe structural events that follow charge transfer to the chromophore at cryogenic temperatures well below the protein dynamical transition (14Vitkup D. Ringe D. Petsko G.A. Karplus M. Nat. Struct. Biol. 2000; 7: 34-38Crossref PubMed Scopus (377) Google Scholar), or on picosecond timescales at ambient temperature. We find the interior of the rigid β-barrel (13Yang F. Moss L.G. Phillips Jr., G.N. Nat. Biotechnol. 1996; 14: 1246-1251Crossref PubMed Scopus (1301) Google Scholar) to be significantly more fluid than the active site of previously characterized photoactive proteins (15Edman K. Nollert P. Royant A. Belrhali H. Pebay-Peyroula E. Hajdu J. Neutze R. Landau E.M. Nature. 1999; 401: 822-826Crossref PubMed Scopus (309) Google Scholar, 16Teng T.Y. Srajer V. Moffat K. Nat. Struct. Biol. 1994; 1: 701-705Crossref PubMed Scopus (194) Google Scholar, 17Schlichting I. Berendzen J. Phillips Jr., G.N. Sweet R.M. Nature. 1994; 371: 808-812Crossref PubMed Scopus (341) Google Scholar), supporting the proposed (18Fenimore P.W. Frauenfelder H. McMahon B.H. Young R.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14408-14413Crossref PubMed Scopus (473) Google Scholar) existence of protein conformational dynamics far below the solvent glass transition near 200 K. We conclude that rapid, low barrier structural dynamics stabilize the locally created buried charge and account for the bright fluorescence of GFP. Crystallization and Phototransformation—Wild type GFP was expressed and purified essentially according to a previous study (13Yang F. Moss L.G. Phillips Jr., G.N. Nat. Biotechnol. 1996; 14: 1246-1251Crossref PubMed Scopus (1301) Google Scholar). Crystals were grown at 4 °C in 100 mm Tris/HCl, 50 mm MgCl2, and 15% polyethylene glycol 3350, pH 7.8, and cryo-protected using 20% glycerol, 100 mm Tris/HCl, 50 mm MgCl2, and 15% polyethylene glycol 3350, pH 7.8. For the initial studies, GFPA crystals with various dimensions were exposed to an increasing dosage of short-wavelength UV radiation, and x-ray diffraction data were collected, using an in-house rotating anode source, to 2.4-Å resolution. The of electron of the of Glu-222 was used as an for For these initial were not fully and from rigid and were to the electron changes at Glu-222. The appearance of difference in the at 2.4-Å resolution at resolution the of the lumi state were at 100 K to the phototransformation as well as fluorescence and absorption of crystals after these that with illumination to the of a with a of phototransformation and of a intermediate at 100 K was by illumination with a short-wavelength UV UV = with a of for rotating the The at nm for the was to be using an of X-ray with dimensions of 200 was in in a and at 100 K using an at a of X-ray diffraction data were at at the The was an was and the were to 100 200 a GFPA was to resolution the was and a was for the photoproduct GFPL the was to 170 K with a of and at for 15 for structural The was to 100 K with a of by 50 and the was The exposed to x-ray for more than a single of was by the and the between was to be The data and were for that and for the resolution are after the for the resolution are after the for the resolution are after the for the resolution are after the of the for the of were in the GFPA and to the GFPL and for the resolution are after the of the for the of were in the GFPA and to the GFPL and in a and were with on Scholar) and with 4 Biol. 1994; PubMed Scopus Google Scholar). of the were for and the was in the and high was with 4 Biol. 1994; PubMed Scopus Google Scholar), using as a was using and the were with 4 Biol. 1994; PubMed Scopus Google Scholar) and A. E. for of The Scholar) using of chromophore in the phenolic and were with The of the 100 K photoproduct and the 170 K annealed diffraction were to the using 4 Biol. 1994; PubMed Scopus Google Scholar), with of and and were to with and common and of and and of and analysis a of = and for and for the of to = and for of to and = and for of to The of the were for resolution and near The were in for the and difference electron were using 4 Biol. 1994; PubMed Scopus Google Scholar), with of a and used to the of the and annealed The and difference in and were using from the GFPA and the GFPL after For water molecules in the GFPL and annealed that are from in the GFPA the was that was as well as in of the difference at an of than and difference electron is significantly from the chromophore, a of the of the protein and between the the strong are more in the T203H/Q69L double mutant was created from an for J. PubMed Scopus Google Scholar), provided by using the for the using and either a GFP at or a GFP in was on an infrared and data were at cm-1 resolution with the by a at the nm of a or the nm of a the in the photogenerated were For were between by a IR difference with wild type GFP were to the A. E. Nat. Biotechnol. 1996; 14: PubMed Scopus Google data in Fig. was used for of to wild type GFP. IR were at the at the M. J. Matousek P. R. N. Phillips D. PubMed Scopus Google Scholar). The 400 nm of a excited a mm of in either or and in a to infrared of the and of an in a type generated a IR with at cm-1 cm-1 at which was and The IR and 400 nm excitation were the to The was with resolution in a with a and and with to the are between of in the and of the excitation by the with to the excitation The response was 400 and as by the absorption of the GFPA group of the crystals was with in the and between the and were at The that an that is on to (12van Thor J.J. Gensch T. Hellingwerf K.J. Johnson L.N. Nat. Struct. Biol. 2002; 9: 37-41Crossref PubMed Scopus (202) Google Scholar). In the chromophore of the A in with to the of Glu-222 and the electron a structure that is to the wild type structure at resolution at pH (13Yang F. Moss L.G. Phillips Jr., G.N. Nat. Biotechnol. 1996; 14: 1246-1251Crossref PubMed Scopus (1301) Google Scholar). In particular, the Glu-222 is not in a is and with the = = and the electron of the (11van Thor J.J. Zanetti G. Ronayne K. Towrie M. J. Phys. Chem. B. 2005; 109: 16099-16108Crossref PubMed Scopus (62) Google Scholar). This with the wild type structure at resolution at pH Glu-222 was with and (7Brejc K. Sixma T.K. Kitts P.A. Kain S.R. Tsien R.Y. Ormo M. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2306-2311Crossref PubMed Scopus (654) Google in GFPA is of an of as in the low pH structure (7Brejc K. Sixma T.K. Kitts P.A. Kain S.R. Tsien R.Y. Ormo M. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2306-2311Crossref PubMed Scopus (654) Google Scholar). X-ray and IR of the 100 K GFPL with cryogenically intermediates of photoactive proteins (15Edman K. Nollert P. Royant A. Belrhali H. Pebay-Peyroula E. Hajdu J. Neutze R. Landau E.M. Nature. 1999; 401: 822-826Crossref PubMed Scopus (309) Google Scholar, 16Teng T.Y. Srajer V. Moffat K. Nat. Struct. Biol. 1994; 1: 701-705Crossref PubMed Scopus (194) Google Scholar, 17Schlichting I. Berendzen J. Phillips Jr., G.N. Sweet R.M. Nature. 1994; 371: 808-812Crossref PubMed Scopus (341) Google Scholar), and strong structural changes at 100 K are from the difference electron The of in resolution the between and in the resolution the of with increasing and of the resolution the and low of the strong electron in the difference Strong in of the and of Glu-222 in resulting from the and light-induced decarboxylation of in crystals of GFP. charge transfer from Glu-222 to the chromophore conformational changes in the chromophore and and difference active site in GFPL than in GFPA The changes include perturbation of the which from by the The more and the from the to the oxygen by Å in the with the difference changes these structural to chromophore ionization in both crystals and The of the absorption and changes in the cm-1 of the GFPL GFPA IR difference previously to ionization of the chromophore Thor J.J. I. A. Hellingwerf K.J. 1998; PubMed Scopus Google Scholar). In particular, and on a for the GFP chromophore Tonge P.J. 2002; 4: PubMed Scopus Google Scholar) of with of the group and the and the of the chromophore in the IR difference (11van Thor J.J. Zanetti G. Ronayne K. Towrie M. J. Phys. Chem. B. 2005; 109: 16099-16108Crossref PubMed Scopus (62) Google Scholar, Thor J.J. I. A. Hellingwerf K.J. 1998; PubMed Scopus Google Scholar). We identify a and at 1697 and cm-1 with a perturbation of the of Gln-69, on A. Biophys. Mol. Biol. 2000; PubMed Scopus Google Scholar) and the of in the phototransformed mutant We that the double mutant be in to the single for which the chromophore is in equilibrium S. Google Scholar). of a cm-1 is with of the hydrogen in The appearance of suggests for the photogenerated CO2. with the dominant to the difference electron the of the molecule the of the chromophore environment at 100 K. IR data on single crystals J. J. and J. T. in the of the hydrogen and a of with distinct and to in the chromophore to the difference by include an water and for A of which hydrogen to the of Gln-69, and Glu-222, to the of and to of the chromophore to a in the fully relaxed photoproduct state (12van Thor J.J. Gensch T. Hellingwerf K.J. Johnson L.N. Nat. Struct. Biol. 2002; 9: 37-41Crossref PubMed Scopus (202) Google Scholar). The GFPL GFPA IR difference reveals in a for The of these to stabilize the chromophore more than in at 100 K that of the GFPL GFPA IR difference with from processes at temperature. IR including the appearance of from illumination at either or The the of with the of reduction in the phototransformation quantum yield (12van Thor J.J. Gensch T. Hellingwerf K.J. Johnson L.N. Nat. Struct. Biol. 2002; 9: 37-41Crossref PubMed Scopus (202) Google Scholar) at The of phototransformation of GFP at 100 K is of below that to of in at cryogenic structural changes of the are not difference K. J. McMahon B.H. Sweet R.M. Berendzen J. I. Nature. 2000; PubMed Scopus Google Scholar). of GFP not the for the excited chromophore to by the UV is on the of and experimental using a as is from x-ray absorption S. M. Biol. PubMed Scopus Google Scholar). X-ray and IR of the structure in a GFPL that is from 100 to 170 at for 15 and to 100 K. The resulting difference electron reveals structural changes the the including the formation of a of structural in the chromophore A of and of the to of the and the of the in the The with within hydrogen bonding of the chromophore was previously in (12van Thor J.J. Gensch T. Hellingwerf K.J. Johnson L.N. Nat. Struct. Biol. 2002; 9: 37-41Crossref PubMed Scopus (202) Google Scholar) as well as in the mutant an chromophore M. K. Tsien R.Y. Remington S.J. 1996; PubMed Scopus Google Scholar) and with the of at in the GFPL of the intermediate produced by phototransformation at 200 K (λmax = nm) are intermediate between for the GFPL and A and of the chromophore environment following ionization is from the of with to in a phenolate at and cm-1 at 100 and The cm-1 of the light-induced GFPA difference at 200 K the GFPA difference at ambient than the GFPL GFPA at 100 K However, the of the 1697 cm-1 to and the the formation of a distinct intermediate state at 200 K. The IR to and to transition to the state at the protein fully relax to accommodate the stress by The exhibits in the chromophore The the K a single band at cm-1 at 200 K the difference the with in the a water molecule site in the or electron be to the molecule The of solvent in the active site suggests that the and are and that the pathway is strong for a well water in GFPA and GFPL that structural annealing the or of IR a Structural in the and ESPT for the fluorescence photocycle (7Brejc K. Sixma T.K. Kitts P.A. Kain S.R. Tsien R.Y. Ormo M. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2306-2311Crossref PubMed Scopus (654) Google Scholar, 8Palm G.J. Zdanov A. Gaitanaris G.A. Stauber R. Pavlakis G.N. Wlodawer A. Nat. Struct. Biol. 1997; 4: 361-365Crossref PubMed Scopus (235) Google Scholar) an intermediate structure I* with an electrostatic environment that following the chromophore is and the proposed Glu-222 proton acceptor is time-resolved infrared difference absorption on GFP 50 after 400 nm excitation of the neutral chromophore a at in to the 1697 cm-1 The cm-1 for of a neutral B. J. A. Biophys. J. 2005; 88: Full Text Full Text PDF PubMed Scopus Google Scholar), and cm-1 with Glu-222 as the proton residues are exposed to solvent and are to be fully in equilibrium at pH This evidence is in with a (9Stoner-Ma D. Jaye A.A. Matousek P. Towrie M. Meech S.R. Tonge P.J. J. Am. Chem. Soc. 2005; 127: 2864-2865Crossref PubMed Scopus (170) Google Scholar, Thor J.J. Zanetti G. Ronayne K. Towrie M. J. Phys. Chem. B. 2005; 109: 16099-16108Crossref PubMed Scopus (62) Google Scholar). from transient infrared on the E222D mutant (11van Thor J.J. Zanetti G. Ronayne K. Towrie M. J. Phys. Chem. B. 2005; 109: 16099-16108Crossref PubMed Scopus (62) Google Scholar). cm-1 and 1697 cm-1 are after excitation, rapid proton transfer to Glu-222 and structural perturbation of Gln-69. We the structural and spectroscopic changes that in the phototransformation pathway that is by the light-induced decarboxylation of the buried at low a lumi GFPL, that is characterized by strong structural changes to the state GFPA, as by x-ray changes in the that the GFPL state is and difference be to on the of the x-ray the of infrared absorption of phototransformed and In particular, the 1697 cm-1 is to structural perturbation of and the cm-1 band to structural annealing of the GFPL photoproduct the metastable intermediate GFPM, which is characterized by solvent and protein time-resolved transient infrared of the fluorescence that the structural response to excitation is to the response in the phototransformation reaction pathway. The of the 1697 cm-1 band in the GFPL, and intermediates to common structural mechanisms in the fluorescence and phototransformation and suggests that the intermediates in the fluorescence photocycle. This spectroscopic signature the strong structural perturbation of that is in the difference electron Structural perturbations of Cys-70, the solvent and that are in the difference electron are to the fluorescence photocycle as of the electrostatic response to chromophore The absorption of GFPL that of recently state intermediates nm) and nm) in the fluorescence photocycle at M. Thor J.J. R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: PubMed Scopus Google Scholar), which relax to GFPA with a the blue of the transition in the fluorescence pathway is in than the blue in the transition in the phototransformation pathway. to the structure structural including of that are not to in the fluorescence We that the low barrier structural changes are in GFP, the fluorescence photocycle after the initial excited state ionization and competing non-radiative fluid of the chromophore environment are in of proposed in the that the fluorescence of the chromophore H. S. T. T. S. M. M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). picosecond structural of water molecules and in the chromophore environment stabilize the charge in the for the high fluorescence quantum efficiency under blue The low fluorescence quantum of the T203H J. PubMed Scopus Google Scholar) and T203H/Q69L mutants, the interactions between the chromophore and the perturbed are or In the T203H/Q69L mutant the chromophore be from solvent in the the H-bonding network connecting the chromophore with solvent, Gln-69, and be The spectroscopic of the double mutant not a of the non-radiative to be at low changes in structure of wild type GFP stabilize the charge following ionization of the buried chromophore. excited state remains at K a and fluorescence emission at nm (3Chattoraj M. King B.A. Bublitz G.U. Boxer S.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8362-8367Crossref PubMed Scopus (796) Google Scholar). This evidence the existence of low barrier dynamics below the solvent glass transition (18Fenimore P.W. Frauenfelder H. McMahon B.H. Young R.D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14408-14413Crossref PubMed Scopus (473) Google Scholar). We in the photoactive protein, electrostatic rearrangements resulting from proton transfer between the buried and the chromophore phenolic oxygen the protein to A. J. Hellingwerf K.J. 2001; PubMed Scopus Google Scholar). This the electrostatic rearrangements that are the fluorescence of GFP. In GFP, we strong difference electron in to a of protein (15Edman K. Nollert P. Royant A. Belrhali H. Pebay-Peyroula E. Hajdu J. Neutze R. Landau E.M. Nature. 1999; 401: 822-826Crossref PubMed Scopus (309) Google Scholar, 16Teng T.Y. Srajer V. Moffat K. Nat. Struct. Biol. 1994; 1: 701-705Crossref PubMed Scopus (194) Google Scholar, 17Schlichting I. Berendzen J. Phillips Jr., G.N. Sweet R.M. Nature. 1994; 371: 808-812Crossref PubMed Scopus (341) Google Scholar). a study of the reaction strong difference electron with illumination at low N. J. Am. Chem. Soc. 2004; PubMed Scopus Google Scholar). be that in data and data comparison with transfer and charge are in S.J. S. PubMed Scopus Google Scholar). The mechanisms that stabilize the electrostatic changes in GFP, which are proposed to be to the of non-radiative therefore Structural dynamics are in the of quantum S.J. S. PubMed Scopus Google Scholar, J. Biochem. 2002; PubMed Scopus Google Scholar) and electron transfer K. Nature. PubMed Scopus Google Scholar, Nature. 1999; PubMed Scopus Google Scholar). However, these dynamics are not and are from or for in to GFP a well characterized that is to structural dynamics using We that of the GFP photocycle the protein to (1Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4982) Google Scholar, 2Lippincott-Schwartz J. Snapp E. Kenworthy A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 444-456Crossref PubMed Scopus (986) Google Scholar) the high fluorescence In particular, the that in the neutral state which has the fluorescence quantum the hydrogen bonding including and and We on for We of the at the to J. V. and for rapid The GFP crystals used in were with the of We Moffat for of the