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Variations on the GFP Chromophore

2004; Elsevier BV; Volume: 280; Issue: 4 Linguagem: Inglês

10.1074/jbc.c400484200

1083-351X

Pascal G. Wilmann, Jan Petersen, Rodney J. Devenish, Mark Prescott, Jamie Rossjohn,

Cell Image Analysis Techniques

We have determined to 2.1 Å resolution the crystal structure of a dark state, kindling fluorescent protein isolated from the sea anemone, Anemonia sulcata. The chromophore sequence Met63-Tyr64-Gly65 of the A. sulcata chromoprotein was previously proposed to comprise a 6-membered pyrazine-type heterocycle (Martynov, V. I., Savitsky, A. P., Martynova, N. Y., Savitsky, P. A., Lukyanov, K. A., and Lukyanov, S. A. (2001) J. Biol. Chem. 276, 21012–21016). However, our crystallographic data revealed the chromophore to comprise a 5-membered p-hydroxybenzylideneimidazolinone moiety that adopts a non-coplanar trans conformation within the interior of the GFP β-can fold. Unexpectedly, fragmentation of the polypeptide was found to occur within the chromophore moiety, at the bond between Cys62C and Met63N1. Our structural data reveal that fragmentation of the chromophore represents an intrinsic, autocatalytic step toward the formation of the mature chromophore within the specific GFP-like proteins. We have determined to 2.1 Å resolution the crystal structure of a dark state, kindling fluorescent protein isolated from the sea anemone, Anemonia sulcata. The chromophore sequence Met63-Tyr64-Gly65 of the A. sulcata chromoprotein was previously proposed to comprise a 6-membered pyrazine-type heterocycle (Martynov, V. I., Savitsky, A. P., Martynova, N. Y., Savitsky, P. A., Lukyanov, K. A., and Lukyanov, S. A. (2001) J. Biol. Chem. 276, 21012–21016). However, our crystallographic data revealed the chromophore to comprise a 5-membered p-hydroxybenzylideneimidazolinone moiety that adopts a non-coplanar trans conformation within the interior of the GFP β-can fold. Unexpectedly, fragmentation of the polypeptide was found to occur within the chromophore moiety, at the bond between Cys62C and Met63N1. Our structural data reveal that fragmentation of the chromophore represents an intrinsic, autocatalytic step toward the formation of the mature chromophore within the specific GFP-like proteins. The green fluorescent protein (GFP) 1The abbreviations used are: GFP, green fluorescent protein; asCP, chromoprotein isolated from Anemonia sulcata; KFP, kindling fluorescent protein; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; r.m.s., root mean square; HPLC, high pressure liquid chromatography.1The abbreviations used are: GFP, green fluorescent protein; asCP, chromoprotein isolated from Anemonia sulcata; KFP, kindling fluorescent protein; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; r.m.s., root mean square; HPLC, high pressure liquid chromatography. from Aequorea victoria has generated widespread interest as a biotechnological tool (1Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4900) Google Scholar). Protein engineering, together with the isolation of novel homologs of GFP, has provided a range of GFP-like proteins that cover the entire visible spectrum and exhibit a variety of useful properties (1Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4900) Google Scholar, 2Verkhusha V.V. Lukyanov K.A. Nat. Biotechnol. 2004; 22: 289-296Crossref PubMed Scopus (273) Google Scholar). The characterized GFP-like proteins can be divided into two groups, the fluorescent proteins and nonfluorescent or weakly fluorescent chromoproteins (3Labas Y.A. Gurskaya N.G. Yanushevich Y.G. Fradkov A.F. Lukyanov K.A. Lukyanov S.A. Matz M.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4256-4261Crossref PubMed Scopus (297) Google Scholar, 4Matz M.V. Lukyanov K.A. Lukyanov S.A. BioEssays. 2002; 24: 953-959Crossref PubMed Scopus (130) Google Scholar). The chromophore within these proteins arises from a unique autocatalytic post-translational modification of three consecutive amino acids (XYG) in the primary sequence, which, to date, has been observed to form a 5-membered heterocycle. In some GFP-like proteins, the chromophore maybe modified by further post-translational events. For example, some fluorescent proteins and chromoproteins with red-shifted spectral properties have an extended π -conjugation system resulting from acylimine formation; photo-conversion of the Kaede protein and GFP is due to a unique UV-induced polypeptide cleavage and decarboxylation event, respectively (5Mizuno H. Mal T.K. Tong K.I. Ando R. Furuta T. Ikura M. Miyawaki A. Mol. Cell. 2003; 12: 1051-1058Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 6van Thor J.J. Gensch T. Hellingwerf K.J. Johnson L.N. Nat. Struct. Biol. 2002; 9: 37-41Crossref PubMed Scopus (199) Google Scholar). X-ray crystallographic studies have been important in revealing that the chromophore can adopt alternative conformations such as trans non-coplanar and trans-coplanar present in the essentially nonfluorescent chromoprotein (Rtms5) and the far-red fluorescent protein (eqFP611), respectively (7Prescott M. Ling M. Beddoe T. Oakley A.J. Dove S. Hoegh-Guldberg O. Devenish R.J. Rossjohn J. Structure (Camb.). 2003; 11: 275-284Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 8Petersen J. Wilmann P.G. Beddoe T. Oakley A.J. Devenish R.J. Prescott M. Rossjohn J. J. Biol. Chem. 2003; 278: 44626-44631Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The range of possible post-translational modifications of the chromophore was recently extended (9Martynov V.I. Savitsky A.P. Martynova N.Y. Savitsky P.A. Lukyanov K.A. Lukyanov S.A. J. Biol. Chem. 2001; 276: 21012-21016Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) with the proposal of an alternative cyclization mechanism for the chromoprotein asCP, isolated from the sea anemone Anemonia sulcata. The model proposed a unique 6-membered pyrazine-type heterocycle. In addition, analysis by SDS-PAGE indicated that asCP under-went fragmentation at a position adjacent to the chromophore. However, an NMR study of the "chromopeptide" isolated by proteolytic digestion of asFP595, a double point mutant (T68A/A143S) of asCP, suggested the alternative cyclization mechanism to be incorrect, and the protein instead contains a p-hydroxybenzylideneimidazolinone moiety typical of GFP-like proteins (10Zagranichny V.E. Rudenko N.V. Gorokhovatsky A.Y. Zakharov M.V. Balashova T.A. Arseniev A.S. Biochemistry. 2004; 43: 13598-13603Crossref PubMed Scopus (16) Google Scholar). A similar chromophore structure and fragmentation was identified in the highly yellow fluorescent protein zFP538 (11Zagranichny V.E. Rudenko N.V. Gorokhovatsky A.Y. Zakharov M.V. Shenkarev Z.O. Balashova T.A. Arseniev A.S. Biochemistry. 2004; 43: 4764-4772Crossref PubMed Scopus (18) Google Scholar). asCP is a very weakly fluorescent protein that can undergo photoconversion (termed "kindling") to become fluorescent (Emmax 595 nm) (12Lukyanov K.A. Fradkov A.F. Gurskaya N.G. Matz M.V. Labas Y.A. Savitsky A.P. Markelov M.L. Zaraisky A.G. Zhao X. Fang Y. Tan W. Lukyanov S.A. J. Biol. Chem. 2000; 275: 25879-25882Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). It has been proposed that alterations in the configuration of the chromophore form the basis of the kindling phenomenon (13Chudakov D.M. Feofanov A.V. Mudrik N.N. Lukyanov S. Lukyanov K.A. J. Biol. Chem. 2003; 278: 7215-7219Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The kindling properties of asCP have been optimized with the inclusion of the key mutation (A143G) in the vicinity of the chromophore to produce a variant, termed KFP (14Chudakov D.M. Belousov V.V. Zaraisky A.G. Novoselov V.V. Staroverov D.B. Zorov D.B. Lukyanov S. Lukyanov K.A. Nat. Biotechnol. 2003; 21: 191-194Crossref PubMed Scopus (272) Google Scholar). To clarify the controversy in the literature regarding the asCP and zFP538 chromophore conformation, and to begin to gain an understanding of the kindling phenomenon, we determined the 2.1 Å resolution crystal structure of KFP. The structure highlights several key features, including fragmentation of the former bond between Cys62C and Met63N1, and gives a detailed view of the chromophore and its environment indicating the presence of p-hydroxybenzylideneimidazolinone moiety in a trans non-coplanar configuration. Cloning—Using the vector pKindling-Red-N (Evrogen) as a template, a DNA cassette encoding KFP was retrieved by PCR using the oligonucleotide primer pair 5′-taggatccatcgccaccatggtgagcggcctg-3′ and 5′-atagtttagcggccgctcagtgatcagagttggccttctcgggcag-3′ and was cloned into the BamHI/NotI expression site pQE10N to produce pQE10N::KFP (15Beddoe T. Ling M. Dove S. Hoegh-Guldberg O. Devenish R.J. Prescott M. Rossjohn J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 597-599Crossref PubMed Scopus (20) Google Scholar). Protein Expression and Purification—BL21(DE3) cells harboring pQE10N::KFP were induced for protein expression and protein-purified by a combination of nickel-nitriloacetic acid and S200 gel filtration chromatography as described (8Petersen J. Wilmann P.G. Beddoe T. Oakley A.J. Devenish R.J. Prescott M. Rossjohn J. J. Biol. Chem. 2003; 278: 44626-44631Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 15Beddoe T. Ling M. Dove S. Hoegh-Guldberg O. Devenish R.J. Prescott M. Rossjohn J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 597-599Crossref PubMed Scopus (20) Google Scholar). Protein eluting at a position corresponding to the KFP tetramer was concentrated to 20 mg/ml using a 10,000 molecular weight cut-off centrifugal filter unit (Centricon, Millipore) in preparation for crystallization trials. Chromopeptide Isolation and MALDI-TOF MS—KFP (1 mg/ml) was adjusted to pH 2.3 with dilute HCl and immediately readjusted to pH 7.8 with dilute NaOH. Digestion with trypsin (1:50, trypsin:protein; Sigma) was performed for 4 h at room temperature. Digestions were immediately adjusted to 0.1% (v/v) trifluoroacetic acid and applied to an XTerra analytical C18 column (Waters); peptides were eluted with a linear elution gradient of 0.1% (v/v) trifluoroacetic acid in 60% (v/v) acetonitrile and detected by absorbance at 214 nm. Visibly colored fractions containing chromopeptide were collected for further analysis. MALDI-TOF MS analysis of chromopeptide was performed as described (7Prescott M. Ling M. Beddoe T. Oakley A.J. Dove S. Hoegh-Guldberg O. Devenish R.J. Rossjohn J. Structure (Camb.). 2003; 11: 275-284Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Crystallization and Data Collection—Crystals were grown by the hanging drop vapor diffusion method at 20 °C in 16% polyethylene glycol 3350, 0.1 m sodium acetate, pH 5.7, 0.2 m ammonium acetate. Crystals were observed after 3–4 days and grew to a maximal crystal size after 7–14 days. Crystals were flash-cooled to 100 K prior to data collection using 20% polyethylene glycol 3350, 0.1 m sodium acetate, 0.2 m ammonium acetate, and 15% glycerol as the cryoprotectant. X-ray diffraction experiments were performed utilizing a Rikagu RU-3HBR rotating anode generator with helium purged OSMIC focusing mirrors coupled to an R-AXIS IV++ detector. Crystals were determined to belong to space group P6222 with unit cell dimensions of a = b = 112 Å, c = 97 Å, α = β = 90°, γ = 120°. The Matthews' coefficient (17Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7911) Google Scholar) suggested that there is 1 molecule/asymmetric unit with a solvent content of 67%. For a full summary of the data collection statistics refer to Table I.Table IData collection and refinement statisticsKFPpH 5.7Temperature100 KSpace groupP6222Cell dimensions (Å) (a, b, c)112.482, 112.482, 96.906Resolution (Å)50.0 -2.1Total no. of observations176,830No. of unique observations21,515Multiplicity8.2Data completeness (%)98.6 (99.2)aThe values in parentheses are for the highest resolution bin (approximate interval 0.1 Å).No. data > 2 σI78.8 (54.2)I/σI28.6 (3.1)RmergebRmerge = Σ|Ihkl - 〈Ihkl〉|/ΣIhkl. (%)7.1 (60.5)Non-hydrogen atoms Protein1762 Chromophore23 Ion4 Water130Resolution (Å)50.0-2.1RfactorcRfactor = Σhkl ∥Fo| -|Fc∥/Σhkl|Fo| for all data except for 5%, which was used for the Rfree calculation. (%)21.4RfreedRfree calculation. (%)23.3r.m.s. deviations from ideality Bond lengths (Å)0.006 Bond angles (°)1.33 Impropers (°)0.82 Dihedrals (°)26.1Ramachandran plot Most favored region (%)90.9 Allowed region (%)9.1B-factors (Å2) Average main chain43.3 Average side chain44.5 Average water molecule46.8 Chromophore39.6 Acetate Ion43.4 r.m.s. deviation bonded Bs1.69a The values in parentheses are for the highest resolution bin (approximate interval 0.1 Å).b Rmerge = Σ|Ihkl - 〈Ihkl〉|/ΣIhkl.c Rfactor = Σhkl ∥Fo| -|Fc∥/Σhkl|Fo| for all data except for 5%, which was used for the Rfree calculation.d Rfree calculation. Open table in a new tab Structure Determination and Refinement—The crystal structure of KFP was solved by molecular replacement using the program AmoRe in the CCP4 suite. A modified protomer of the eqFP611 structure (Protein Data Bank code 1UIS) was used as the search probe, with the chromophore removed and all sequence differences mutated to alanine. The structure was readily solved with one clear peak in the rotation function leading to the elucidation of one clear peak in the translation function. Refinement was monitored by the Rfree value (5.0% of the data) with neither a sigma nor a low resolution cut-off being applied to the data. The structure was refined using rigid body fitting followed by the simulated annealing protocol of crystallography NMR software (CNS version 1.1) interspersed with rounds of model building using the program O (18Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16948) Google Scholar, 19Jones T.A. Zou J-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar). Restrained individual B-factor refinement was employed, and bulk solvent corrections were applied to the data set. Water molecules were included in the model if they were within hydrogen-bonding distance to chemically reasonable groups, appeared in Fo – Fc maps contoured at 3.5σ, and had a B-factor less than 60 Å2. Clear electron density for the Met63-Tyr64-Gly65 chromophore sequence was observed in the initial 2Fo – Fc and Fo – Fc maps and was built in the penultimate round of refinement. A p-hydroxybenzylideneimidazolinone moiety in a trans non-coplanar conformation fitted into the observed density unambiguously. Attempts to model other chromophore conformations, such as the previously postulated 6-membered pyrazine-type heterocycle (9Martynov V.I. Savitsky A.P. Martynova N.Y. Savitsky P.A. Lukyanov K.A. Lukyanov S.A. J. Biol. Chem. 2001; 276: 21012-21016Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), failed to adequately represent the observed electron density and led to a worsening of the Rfac and Rfree values. Topology and parameter files for the chromophore were initially based on those observed for the eqFP611 chromophore (8Petersen J. Wilmann P.G. Beddoe T. Oakley A.J. Devenish R.J. Prescott M. Rossjohn J. J. Biol. Chem. 2003; 278: 44626-44631Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The linkage between the chromophore and Ser66 was observed to represent a true trans peptide bond, analogous to that observed in the eqFP611, DsRed, and Rtms5 structures. However, a break in the electron density between Cys62 and the chromophore was observed, indicative of cleavage of polypeptide chain at this site. Trying to model a covalent link between Cys62 and the chromophore led to a worsening of the Rfac and Rfree values; by contrast, modeling a covalent break at the same site resulted in an improved fit of the chromophore into the electron density. In addition, simulated annealing omit maps of the chromophore and the surrounding residues were created to validate the correctness of our interpretation of the chromophore structure. The final model, which comprises a single protomer (residues 4–232), 130 water molecules, and 1 acetate ion, has an Rfac of 21.4% and an Rfree of 23.3% for all reflections between 50 and 2.1 Å. See Table I for a summary of model quality and refinement statistics. The structure has been deposited in the Protein Data Bank (code 1XQM). Structure Description—The protomer in the asymmetric unit is very similar to the described previously GFP 11-stranded β-barrel with a central α-helix running co-axially to the barrel axis (Fig. 1A). At the center of the barrel the cyclic tripeptide chromophore is observed to form only one covalent link to the protein, with a break in the polypeptide between Cys62 and the chromophore (Fig. 1, B and C). SDS-PAGE analysis of redissolved KFP crystals as well as KFP starting material revealed polypeptide fragments with molecular masses wholly consistent with the position of fragmentation observed in the crystal structure. Similar results showing fragmentation have been reported for asFP595 (9Martynov V.I. Savitsky A.P. Martynova N.Y. Savitsky P.A. Lukyanov K.A. Lukyanov S.A. J. Biol. Chem. 2001; 276: 21012-21016Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). These results indicate that fragmentation of KFP is a property of the native protein and an intrinsic step in the maturation of the chromophore that must take place inside the native protein fold. In comparison with other GFP-like proteins, KFP is most similar to eqFP611 (59.2% sequence identity, 218 equivalent Cα atoms having an r.m.s. deviation of 0.74 Å) (8Petersen J. Wilmann P.G. Beddoe T. Oakley A.J. Devenish R.J. Prescott M. Rossjohn J. J. Biol. Chem. 2003; 278: 44626-44631Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), DsRed (47.4% sequence identity, 211 equivalent Cα atoms having an r.m.s. deviation of 0.81 Å), and Rtms5 (46.5% sequence identity, 213 equivalent Cα atoms having an r.m.s. deviation of 0.86 Å) (16Wall M.A. Socolich M. Ranganathan R. Nat. Struct. Biol. 2000; 7: 1133-1138Crossref PubMed Scopus (300) Google Scholar), whereas less similarity is observed between KFP and GFP (21.4% sequence identity, 201 equivalent Cα atoms having an r.m.s. deviation of 1.28 Å). Notable structural differences include the immediate environment of the chromophore and the extended C-terminal tail of KFP. The biologically active 222 tetramer generated via the crystallographic symmetry operators exhibited close structural homology to the DsRed, eqFP611, and Rtms5 tetramers (7Prescott M. Ling M. Beddoe T. Oakley A.J. Dove S. Hoegh-Guldberg O. Devenish R.J. Rossjohn J. Structure (Camb.). 2003; 11: 275-284Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 8Petersen J. Wilmann P.G. Beddoe T. Oakley A.J. Devenish R.J. Prescott M. Rossjohn J. J. Biol. Chem. 2003; 278: 44626-44631Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 16Wall M.A. Socolich M. Ranganathan R. Nat. Struct. Biol. 2000; 7: 1133-1138Crossref PubMed Scopus (300) Google Scholar). However, the intersubunit A-B interface of KFP is moderately larger than that of the GFP-like tetramers. This observation is attributable to the extended C-terminal tail of KFP that participates in numerous intersubunit contacts. Chromophore Structure and Environment—A p-hydroxybenzylideneimidazolinone moiety, typical of GFP cyclization, fitted the observed electron density unambiguously (Fig. 1B). A 6-membered pyrazine-type heterocycle, predicted by the alternative cyclization mechanism for asCP, was inconsistent with the observed electron density (Fig. 1, B and C) (9Martynov V.I. Savitsky A.P. Martynova N.Y. Savitsky P.A. Lukyanov K.A. Lukyanov S.A. J. Biol. Chem. 2001; 276: 21012-21016Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The imidazolinone moiety is sandwiched between the turns of the central helix, making several hydrogen bonds and van der Waals interactions with the surrounding side chains. Of the chromophore contacts in the DsRed, Rtms5, eqFP611, and KFP structures, residues Trp90, Arg92, Glu145, and Glu215 were observed to be strictly conserved. Notably, three of these residues (Trp90, Arg92, and Glu215) were found to contact the imidazolinone moiety, highlighting their possible important role in chromophore cyclization (Fig. 2A). The electron density shows clearly that the imidazolinone and 4-hydroxyphenylmethylene moieties of the chromophore are non-coplanar, with the 4-hydroxyphenyl moiety rotated 31° in respect to the plane of the imidazolinone ring (Figs. 1 and 2A). The chromophore adopts a trans non-coplanar conformation, stabilized by 6 hydrogen bonds, 5 water-mediated hydrogen bonds, and numerous van der Waals interactions (Table II, Fig. 2A). The methionine moiety of the KFP chromophore is found to extend into a deep pocket that is surrounded by the side chains of residues Gln39, Met41, Ile199, Gln213, and Glu215. The residues that comprise this pocket are largely conserved in the KFP, DsRed, Rtms5, and eqFP611 structures.Table IIChromophore contactsChromophoreProteinNature of interactionMethionine moiety N1Cys62van der Waals Cα1Ser59, Glu215van der Waals Cβ1Ser59, Cys62, Gln213, Glu215van der Waals Cγ1Gln39, Glu215van der Waals Sδ1Gln39, Gln213, Glu215van der Waals Cϵ3Met41, Gln213van der WaalsImidazolinone moiety C1Thr60, Glu215van der Waals N2Thr60van der WaalsGlu215Oϵ2H-bond N3Thr60OH-bond C2Thr60, Arg92van der Waals Cα2Thr60, Glu215van der Waals O2Arg92Nη2H-bondThr60, Lys67van der Waals4-Hydroxyphenyl-methylene moiety Cβ2His197, Glu215van der Waals Cγ2His197van der Waals Cδ1Thr60, Met160, His197van der Waals Cδ2Arg92, Tyr178, His197van der Waals Cϵ1Ser158, Met160, Leu174, His197van der Waals Cϵ2Arg92, Glu145, Tyr178van der Waals Cζ[ρ]Glu145, Ser158, Leu174van der Waals Oη[ρ]Ser158Oγ[ρ]H-bondLeu174van der WaalsGlu145Oϵ2, Thr176Oγ1Water-mediated H-bondGlycyl moiety Cα3Thr60, Trp90van der Waals CLys67, Trp90van der Waals OTrp90Nϵ1, Gln106Nϵ2H-bondSer61O, Thr108Oγ1, Lys67NWater-mediated H-bond Open table in a new tab Fragmentation of KFP results in the formation of a unique chromophore and thus distinguishes it from those observed in other available crystal structures of GFP-like proteins; however the mechanism for fragmentation is not clear. Although the same three amino acids, Met-Tyr-Gly, comprise the chromophore in both KFP and the highly fluorescent eqFP611, the extended conjugation due to the acylimine in eqFP611 is absent in KFP. The cleavage of the Cys62-chromophore bond provides a degree of freedom of movement of the chromophore, permitting a significant shift of the chromophore body toward β-strand 8, which also causes a shift in the position of this strand (as highlighted by the comparison of KFP to the eqFP611 structures (Fig. 2B)). Both the KFP and eqFP611 4-hydroxyphenylmethylene moieties form polar interactions with residue Ser158 despite differences in the position and rotamer conformation of Ser 158. The KFP 4-hydroxyphenylmethylene moiety makes additional interactions through 2 water-mediated hydrogen bonds to Glu145 and Thr176. The hydroxyphenyl moiety of the KFP chromophore makes van der Waals contacts with Thr60, Arg92, Glu145, Ser158, Met160, Leu174, Tyr178, His197, and Glu215 (Table II). The imidazole ring of His197 interacts extensively with the hydroxyphenyl moiety, adopting a coplanar conformation with respect to this moiety. Notably, KFP lacks an aromatic residue at position 174, which in eqFP611 is occupied by a Phe and was observed to make several contacts to the p-hydroxyphenylmethylene moiety. The interplay between residues at positions 197 and 67 observed in DsRed, eqFP611, and the Rtms5 structures is also observed in KFP, where, because of the bulky His197 imidazole ring, Lys67 is pushed away from the hydroxyphenyl moiety to salt bridge to Glu195 and Glu145. The site of fragmentation and chemical nature of the KFP chromophore were investigated in further detail. A peptide released by trypsin digestion of acid denatured KFP (the chromophore is covalently linked to KFP via Ser66) was HPLC-purified and analyzed by MALDI-TOF MS. A 564.20-Da species was observed matching closely the predicted monoisotropic mass (564.26 Da; data not shown) for a pentapeptide (Met-Tyr-Gly-Ser-Lys), which contains the chromophore sequence (Met-Tyr-Gly) and corresponds to a typical GFP-like cyclization event to form the p-hydroxybenzylideneimidazalinone moiety followed by fragmentation to yield the "imino"-substituted chromophore, as predicted for asFP595 and zFP538 (10Zagranichny V.E. Rudenko N.V. Gorokhovatsky A.Y. Zakharov M.V. Balashova T.A. Arseniev A.S. Biochemistry. 2004; 43: 13598-13603Crossref PubMed Scopus (16) Google Scholar, 11Zagranichny V.E. Rudenko N.V. Gorokhovatsky A.Y. Zakharov M.V. Shenkarev Z.O. Balashova T.A. Arseniev A.S. Biochemistry. 2004; 43: 4764-4772Crossref PubMed Scopus (18) Google Scholar). The predicted monoisotropic mass for the "keto"-substituted chromopeptide (a predicted hydrolysis product of the imino form (10Zagranichny V.E. Rudenko N.V. Gorokhovatsky A.Y. Zakharov M.V. Balashova T.A. Arseniev A.S. Biochemistry. 2004; 43: 13598-13603Crossref PubMed Scopus (16) Google Scholar)) or the 6-membered pyrazine-type heterocycle resulting from the alternative cyclization model (9Martynov V.I. Savitsky A.P. Martynova N.Y. Savitsky P.A. Lukyanov K.A. Lukyanov S.A. J. Biol. Chem. 2001; 276: 21012-21016Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) is 565.26 Da. Accordingly, our data discount these alternatives. The chromophore structure observed in KFP represents another alternative for generating red-shifted spectral properties in GFP-like proteins; whereas an acylimine extends the conjugation system of DsRed (λmax 558 nm) (20Gross L.A. Baird G.S. Hoffman R.C. Baldridge K.K. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11990-11995Crossref PubMed Scopus (519) Google Scholar) and eqFP611 (λmax 559 nm) (21Wiedenmann J. Schenk A. Rocker C. Girod A. Spindler K.D. Nienhaus G.U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11646-11651Crossref PubMed Scopus (212) Google Scholar), an acylimine is absent KFP (λmax 545 nm). In KFP the single π -bond of the imino (C=NH) substituent extends the GFP-chromophore and (together with His197) appears sufficient to promote the red-shifted property of KFP. This suggests that in DsRed and eqFP611 the acyl (C=O) part of the acylimine does not necessarily participate in the delocalization of the chromophore π -system. Accordingly in the structures of DsRed, eqFP611, and Rtms5 this bond is out of plane in respect to the imidazolinone moiety of the chromophore. The soluble protein used to generate KFP crystals was not exposed to the intense excitation illumination required to induce the phenomenon of kindling. The absorbance and fluorescence spectra of KFP from redissolved crystals were similar to the soluble starting material (data not shown). Our structure, therefore, represents the nonkindled or "dark" state of the protein. A model for the kindling mechanism has been proposed (13Chudakov D.M. Feofanov A.V. Mudrik N.N. Lukyanov S. Lukyanov K.A. J. Biol. Chem. 2003; 278: 7215-7219Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In this model intense illumination with green light induces a significant alteration in the configuration of the chromophore from a trans nonfluorescent to a cis fluorescent form. The chromophore in KFP adopts a trans non-coplanar configuration similar to the chromophore configuration found in another nonfluorescent chromoprotein, Rtms5 (7Prescott M. Ling M. Beddoe T. Oakley A.J. Dove S. Hoegh-Guldberg O. Devenish R.J. Rossjohn J. Structure (Camb.). 2003; 11: 275-284Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The A143G substitution presumably permits a greater degree of conformational freedom that allows the chromophore to adopt a cis coplanar conformation in the kindled state. Fragmentation may lower activation barriers for conformational transitions and/or allow the chromophore to assume an alternative conformation. Nevertheless, there appears to be ample room to accommodate the tyrosyl moiety if it should adopt a cis coplanar configuration upon kindling. Further investigation of the mechanism of kindling requires the crystal structure of the photoconverted protein. In conclusion, this crystal structure of KFP shows for the first time fragmentation of the main chain polypeptide for the GFP-like family, revealing an additional layer of complexity in the chromophore chemistry of these GFP-like proteins. Furthermore, it provides unequivocal evidence that fragmentation in the native protein contributes to the formation of a novel imino-substituted chromophore. We thank S. Harris for assistance in mass spectrometry analysis and Peter Gräber for support.