Structural Characterization of a Blue Chromoprotein and Its Yellow Mutant from the Sea Anemone Cnidopus Japonicus
2006; Elsevier BV; Volume: 281; Issue: 49 Linguagem: Inglês
10.1074/jbc.m606921200
ISSN1083-351X
AutoresMitchell C.Y. Chan, S. Karasawa, Hideaki Mizuno, Ivan Bosanac, D.N. Ho, Gilbert G. Privé, Atsushi Miyawaki, Mitsuhiko Ikura,
Tópico(s)Cell Image Analysis Techniques
ResumoGreen fluorescent protein (GFP) and its relatives (GFP protein family) have been isolated from marine organisms such as jellyfish and corals that belong to the phylum Cnidaria (stinging aquatic invertebrates). They are intrinsically fluorescent proteins. In search of new members of the family of green fluorescent protein family, we identified a non-fluorescent chromoprotein from the Cnidopus japonicus species of sea anemone that possesses 45% sequence identity to dsRed (a red fluorescent protein). This newly identified blue color protein has an absorbance maximum of 610 nm and is hereafter referred to as cjBlue. Determination of the cjBlue 1.8 Å crystal structure revealed a chromophore comprised of Gln63-Tyr64-Gly65. The ring stacking between Tyr64 and His197 stabilized the cjBlue trans chromophore conformation along the Cα2-Cβ2 bond of 5-[(4-hydroxyphenyl)methylene]-imidazolinone, which closely resembled that of the "Kindling Fluorescent Protein" and Rtms5. Replacement of Tyr64 with Leu in wild-type cjBlue produced a visible color change from blue to yellow with a new absorbance maximum of 417 nm. Interestingly, the crystal structure of the yellow mutant Y64L revealed two His197 imidazole ring orientations, suggesting a flip-flop interconversion between the two conformations in solution. We conclude that the dynamics and structure of the chromophore are both essential for the optical appearance of these color proteins. Green fluorescent protein (GFP) and its relatives (GFP protein family) have been isolated from marine organisms such as jellyfish and corals that belong to the phylum Cnidaria (stinging aquatic invertebrates). They are intrinsically fluorescent proteins. In search of new members of the family of green fluorescent protein family, we identified a non-fluorescent chromoprotein from the Cnidopus japonicus species of sea anemone that possesses 45% sequence identity to dsRed (a red fluorescent protein). This newly identified blue color protein has an absorbance maximum of 610 nm and is hereafter referred to as cjBlue. Determination of the cjBlue 1.8 Å crystal structure revealed a chromophore comprised of Gln63-Tyr64-Gly65. The ring stacking between Tyr64 and His197 stabilized the cjBlue trans chromophore conformation along the Cα2-Cβ2 bond of 5-[(4-hydroxyphenyl)methylene]-imidazolinone, which closely resembled that of the "Kindling Fluorescent Protein" and Rtms5. Replacement of Tyr64 with Leu in wild-type cjBlue produced a visible color change from blue to yellow with a new absorbance maximum of 417 nm. Interestingly, the crystal structure of the yellow mutant Y64L revealed two His197 imidazole ring orientations, suggesting a flip-flop interconversion between the two conformations in solution. We conclude that the dynamics and structure of the chromophore are both essential for the optical appearance of these color proteins. The green fluorescent protein (GFP) 4The abbreviations used are: GFP, green fluorescent protein; KFP, kindling fluorescent protein; FP, fluorescent protein; CP, chromoprotein; TCEP, Tris(2-carboxyethyl)phosphine. from Aequorea victoria has gained widespread interest as a biological reporter in living cells (1Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4982) Google Scholar). Since its discovery, considerable efforts have been devoted to protein engineering, in conjunction with isolation of new GFP homologs, to expand the visible spectrum and properties of GFP protein family (1Tsien R.Y. Annu. Rev. Biochem. 1998; 67: 509-544Crossref PubMed Scopus (4982) Google Scholar, 2Verkhusha V.V. Chudakov D.M. Gurskaya N.G. Lukyanov S. Lukyanov K.A. Chem. Biol. 2004; 11: 845-854Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Characterized GFP protein family can be divided into two groups, the fluorescent proteins (FPs) and the non-fluorescent chromoproteins (CPs) (3Matz M.V. Lukyanov K.A. Lukyanov S.A. BioEssays. 2002; 24: 953-959Crossref PubMed Scopus (130) Google Scholar, 4Labas 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 (302) Google Scholar). The GFP chromophore arises through a unique autocatalytic post-translational modification of a tripeptide, usually X-Tyr-Gly, in the primary sequence. The conformation and interaction of the chromophore with its local environment determines the spectral properties of the protein. X-ray crystallographic studies (5Quillin M.L. Anstrom D.M. Shu X. O'Leary S. Kallio K. Chudakov D.M. Remington S.J. Biochemistry. 2005; 44: 5774-5787Crossref PubMed Scopus (142) Google Scholar, 7Petersen 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) have revealed the general relationship between the trans non-co-planarity of chromophores found in CPs and the cis co-planarity of chromophores found in FPs, with the exception of eqFP611, which has a trans co-planar chromophore. To date, four CPs from the Anthozoan species have been characterized: Rtms5 from Montipora efflorescens (8Beddoe 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), gtCP from Goniopora tenuidens (9Martynov V.I. Maksimov B.I. Martynova N.Y. Pakhomov A.A. Gurskaya N.G. Lukyanov S.A. J. Biol. Chem. 2003; 278: 46288-46292Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), aeCP597 from Actinia equine (10Shkrob M.A. Yanushevich Y.G. Chudakov D.M. Gurskaya N.G. Labas Y.A. Poponov S.Y. Mudrik N.N. Lukyanov S. Lukyanov K.A. Biochem. J. 2005; 392: 649-654Crossref PubMed Scopus (80) Google Scholar), and asFP595 from Anemonia sulcata (KFP) (5Quillin M.L. Anstrom D.M. Shu X. O'Leary S. Kallio K. Chudakov D.M. Remington S.J. Biochemistry. 2005; 44: 5774-5787Crossref PubMed Scopus (142) Google Scholar). Three-dimensional structures of Rtms5 (6Prescott 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) and KFP (5Quillin M.L. Anstrom D.M. Shu X. O'Leary S. Kallio K. Chudakov D.M. Remington S.J. Biochemistry. 2005; 44: 5774-5787Crossref PubMed Scopus (142) Google Scholar) have been solved previously, both of which show the same fold as GFP and contain an internal chromophore. Studied CPs have exhibited absorbance maxima in a confined range of 560-597 nm (11Pollok B.A. Heim R. Trends Cell Biol. 1999; 9: 57-60Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 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); no CP has been thus far found to absorb at absorbance maxima greater than 600 nm. Here we present a new CP from the Cnidopus japonicus sea anemone species, which absorbs at 610 nm. We report the molecular cloning, characterization and structure determination of this blue CP, hereafter termed cjBlue. We have also generated a yellow mutant variant from cjBlue with a single mutation at the 64 position (Tyr to Leu) using semi-random mutagenesis. We discuss the structural basis for the blue chromophore formation of wild-type cjBlue and for the blue-to-yellow shift in cjBlue(Y64L) mutant, which lacks an aromatic amino acid in the tripeptide chromophore. Elucidation of the structural details of cjBlue and cjBlue(Y64L) helps to answer not only why cjBlue and cjBlue(Y64L) absorb different colors, but also contributes to our better understanding of why FPs can fluoresce having a structural architecture similar to that of CPs. cDNA Cloning and Gene Construction—A sample of the C. japonicus was acquired from the ocean near the Uozu city in Toyama. Total RNA was isolated from the sea anemone by guanidine thiocyanate extraction. Synthesis, amplification using degenerate primers, and generation of full-length cDNA were performed as described previously (23Karasawa S. Araki T. Yamamoto-Hino M. Miyawaki A. J. Biol. Chem. 2003; 278: 34167-34171Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) using the following degenerate primers: 5′-GAAGGRTGYGTCAAYGGRCAY-3′ and 5′-ACVGGDCCATYDGVAAGAAARTT-3′ (R = Arg or Gly; Y = Cys or Thr; V = Arg, Cys, or Gly; D = Arg, Gly, or Thr). The cDNA encoding the protein-coding region was amplified using primers containing 5′ BamHI and 3′ EcoRI sites. The digested product was then cloned in-frame into the BamHI/EcoRI sites of pRSETB (Invitrogen) for bacterial expression. Site-directed and semi-random mutations were introduced as described (19Sawano A. Miyawaki A. Nucleic Acids Res. 2000; 28: E78Crossref PubMed Scopus (303) Google Scholar). Protein Expression and Purification—CjBlue and cjBlue-(Y64L) were subcloned into a pET28a expression vector. Seleno-l-methionine-labeled protein was produced using minimal M9 media and expressed with N-terminal His6 tag using B834 (DE3) Escherichia coli strain (Novagen). Cells were grown in a shaker incubator at 37 °C until an A600 of 1.20 was reached. Upon induction with 1 mm isopropyl 1-thio-β-d-galactopyranoside, the temperature was lowered to 20 °C, and the protein was allowed to express for 48 h. Protein containing the N-terminal polyhistidine tag was purified using nickel-nitrilotriacetic acid resin (Qiagen). The N-terminal His6 tag was subsequently removed using thrombin followed by size exclusion chromatography (Superdex 75, Amersham Biosciences) to achieve satisfactory levels of purity. Incorporation seleno-l-methionine was confirmed via electrospray mass spectrometry. The purified protein was concentrated to 25 mg/ml in a crystallization buffer (20 mm Tris-HCl (pH 7.5), 150 mm NaCl, and 2 mm Tris(2-carboxyethyl)phosphine (TCEP)). Structure Determination—The protein solution was concentrated to 25 mg/ml in Tris-HCl (pH 7.5) with 150 mm NaCl and 2 mm Tris(2-carboxethyl)phosphine (TCEP). Blue plated crystals (P21), with approximate dimensions of 0.4 × 0.3 × 0.08 mm, were grown in hanging drops containing 2 μl of mother liquor at 22 °C for 2 days. The mother liquor contained 0.2 m NaH2PO4, 20 mm Tris-HCl (pH 7.5), 150 mm NaCl, 20% polyethylene 3350, 20% glycerol, and 2 mm TCEP. The crystals were micro-seeded for two rounds, cryo-protected in 20% glycerol, and flashed cooled to 100 K in a stream of nitrogen gas. The cjBlue SAD (Single Anomalous Dispersion) data were collected at 19-ID beamline at the Advanced Photon Source synchrotron facility and were processed with HKL2000. Data were collected at 0.5° oscillation in 30 s exposures and to 99.3% completeness at 1.8 Å. The crystal belongs to the P21 space group with cell dimensions a = 73.86 Å, b = 126.85 Å, c = 100.51 Å, β = 102.10, and with 8 molecules/asymmetric unit. The cjBlue(Y64L) native data were collected in-house on the Bruker X8 PROTEUM. 0.5° oscillations were collected in 45 s exposures. Data were collected to 100% completeness at 2.0 Å. Unit cell dimensions were: a = 74.08 Å, b = 126.93 Å, c = 100.08 Å, and β = 101.97. The cjBlue structure served as a search model for cjBlue(Y64L) in molecular replacement with the chromophore removed to minimize bias. Both structure refinements were performed using crystallography NMR software 1.1. Spectroscopy—The cjBlue, cjBlue(Y64L), cjBlue(H197S), and cjBlue(Y64L/H197S) samples from their respective stock solutions were exchanged into buffer containing 20 mm NaHPO4 and 150 mm NaCl to measure each individual absorbance spectrum. Concentration of each sample was diluted from the stock 25 mg/ml solution to 1 mg/ml. 0.5 ml of diluted sample was gently transferred into a 1-cm path length quartz cuvette. Absorbance was measured using an Ultrospec 2000 UV-visible spectrometer (GE Healthcare). An absorbance scan was initiated from 250 to 750 nm at 1-nm increments. cDNA Isolation and Protein Purification—Degenerate primers were employed to amplify cDNAs isolated from the sea anemone, C. japonicus. The primers covered several conserved amino acid sequences identified from among GFP-like fluorescent proteins found in other Anthozoa species. The missing 5′ and 3′ ends of the cDNA fragment were amplified using the rapid amplification of cDNA ends strategy. The resultant open reading frame coded 225 amino acids with high sequence homologies to KFP (5Quillin M.L. Anstrom D.M. Shu X. O'Leary S. Kallio K. Chudakov D.M. Remington S.J. Biochemistry. 2005; 44: 5774-5787Crossref PubMed Scopus (142) Google Scholar), hcCP (15Remington S.J. Wachter R.M. Yarbrough D.K. Branchaud B. Anderson D.C. Kallio K. Lukyanov K.A. Biochemistry. 2005; 44: 202-212Crossref PubMed Scopus (124) Google Scholar), eqFP611 (7Petersen 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), and Rtms5 (6Prescott 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) (Fig. 1). The full-length protein was expressed in E. coli with a His6 tag at the N terminus and purified using metal affinity chromatography. The protein referred as cjBlue is a chromoprotein, which does not fluoresce but is dark green in color. CjBlue at pH 7.4 displayed a major absorption wavelength maximum at 610 nm (ϵ = 66,700 m-1·cm-1) (Fig. 2) showing the longest wavelength absorption of all known CPs. The absorption spectrum of cjBlue showed high resistance to acidity (up to pH 4) (data not shown).FIGURE 2Absorbance spectra of cjBlue and mutant variants. A pH of 7.0, a temperature of 22 °C, and the mature form of CPs were used to standardize the absorbance profile. Wild-type (WT) cjBlue is shown in blue, cjBlue(Y64L) in yellow, cjBlue(H197S) in green, and cjBlue(Y64L/H197S) in purple.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Structure of cjBlue—The crystal structure of cjBlue, solved by SAD, was determined to 1.8 Å. The cjBlue crystal belongs to the P21 space group. A final Rfactor and Rfree of 19.5 and 22.5%, respectively, were reached (Table 1). The final cjBlue model contained eight subunits (residues 5-232), 16 PO4, and 1330 water molecules in an asymmetric unit. Further, the tertiary structure of cjBlue shares a similar fold to GFP (16Ormo M. Cubitt A.B. Kallio K. Gross L.A. Tsien R.Y. Remington S.J. Science. 1996; 273: 1392-1395Crossref PubMed Scopus (1934) Google Scholar), dsRed (17Wall M.A. Socolich M. Ranganathan R. Nat. Struct. Biol. 2000; 7: 1133-1138Crossref PubMed Scopus (302) Google Scholar), KFP (5Quillin M.L. Anstrom D.M. Shu X. O'Leary S. Kallio K. Chudakov D.M. Remington S.J. Biochemistry. 2005; 44: 5774-5787Crossref PubMed Scopus (142) Google Scholar), and Rtms5 (6Prescott 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). Each subunit consists of β-strands 11 forming a β-barrel with a central helix running co-axially with the axes of the β-barrel (Fig. 3). The central helix of cjBlue connects the chromophore to the rest of the protein (Fig. 3).TABLE 1Data collection and refinement statistics for cjBlue and cjBlue(Y64L)Data collectionSample/Data setWavelengthResolutionReflections (total/unique)CompletenessRmergeaRmerge = ΣhΣi|Ihi – 〈Ih 〉|/ΣhΣi| 〈Ih 〉| × 100, where I is the intensity of i observations for reflection h and Ih is the mean intensity of the reflection〈I/ 〉 〈σ(I) 〉b〈I 〉 / 〈σI 〉, mean intensity/mean standard deviationÅÅ%%cjBlue (Peak)0.979350.0–1.68,835,277/335,34699.3 (100)7.7 (26.1)12.0 (9.0)cjBlue(Y64L) (Native)1.541950.0–1.81,054,020/121,926100 (100)6.2 (38.1)39.65 (3.0)Refinement statisticsSample/Data setResolutionReflections (working/test)Total number of atomsRcryscRcryst = 100 × Σ|Fobs – Fcalc| /Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factor magnitudes, respectively / RfreedRfree as for Rcryst except calculated for 4.7% of the reflections not used for model refinement〈B 〉 Valuer.m.s.er.m.s., root mean square deviationsBondAngle (°)ÅÅ2ÅcjBlue (Peak)50.0–1.8318,579/16,76715,73019.5/22.519.50.0061.673cjBlue(Y64L) (Native)50.0–2.0107,910/5,76115,70619.8/24.416.20.0061.624a Rmerge = ΣhΣi|Ihi – 〈Ih 〉|/ΣhΣi| 〈Ih 〉| × 100, where I is the intensity of i observations for reflection h and Ih is the mean intensity of the reflectionb 〈I 〉 / 〈σI 〉, mean intensity/mean standard deviationc Rcryst = 100 × Σ|Fobs – Fcalc| /Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factor magnitudes, respectivelyd Rfree as for Rcryst except calculated for 4.7% of the reflections not used for model refinemente r.m.s., root mean square Open table in a new tab The crystal structure of cjBlue revealed a pair of four chemically identical subunits that were intimately in contact with one another (Fig. 3A), in a manner similar to that observed for eqFP611 (7Petersen 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), Rtms5 (6Prescott 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), and dsRed (17Wall M.A. Socolich M. Ranganathan R. Nat. Struct. Biol. 2000; 7: 1133-1138Crossref PubMed Scopus (302) Google Scholar). This molecular association is stabilized by 1) inter-barrel interface interactions and 2) association of the C terminus tails with β-strands 11 of adjacent pair of subunits (Fig. 3). Asn20, Asn21, Thr103, Gln105, and Arg179 are residues located at the inter-barrel interface forming hydrogen bonds between subunits. The C-terminal tail interaction involves hydrogen bonding between Ser227, His231, and Asn232 at one tail and Asp196, Arg198, Glu200, and Thr224 within β-strands 11 of an adjacent pair of subunit. The cjBlue chromophore consists of a 5-[(4-hydroxyphenyl)-methylene]-imidazolinone group with an acylimine bond found between the Cα and nitrogen atom positioned at Gln63 (Figs. 3 and 4) (6Prescott 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 additional double bond at Gln63 is comparable with the double bond found at Gln66 of dsRed (17Wall M.A. Socolich M. Ranganathan R. Nat. Struct. Biol. 2000; 7: 1133-1138Crossref PubMed Scopus (302) Google Scholar, 18Yarbrough D. Wachter R.M. Kallio K. Matz M.V. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 462-467Crossref PubMed Scopus (397) Google Scholar). Like other FPs and CPs, the cjBlue chromophore is buried deeply within the β-barrel (Fig. 3) with substituents hydrogen bonded to neighboring residues and internal water molecules. These residues include Thr158 (2.5 Å), Glu145 (3.1 Å), and water molecule W1 (3.3 Å), which are hydrogen bonded to the oxygen of the p-hydroxyphenyl ring (Fig. 4). Remarkably, water molecules near the cjBlue chromophore were all found in the same positions as those seen in the crystal structure of Rtms5 (6Prescott 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 imidazole ring of His197, located near the chromophore, shows a stacking orientation with the p-hydroxyphenyl ring of ∼3.9 Å. The cjBlue chromophore adopts a trans conformation between the Cα2-Cβ2 bond with noticeable bond angle distortion. Because of internal steric hindrance between O2 and Cδ2-H, an increase in the C2-Cα2-Cβ2 bond angle (141°) and the Cα2-Cβ2-Cγ2 bond angle (117°) was observed. Cys145 also appears to contribute to the trans conformation by sterically hindering the p-hydroxyphenyl ring from adopting a cis conformation. Chromophores adopting a trans conformation, with the exception of eqFP611, lack planarity between the imidazolinone and p-hydroxyphenyl ring because of internal collision of O2 and Cδ2-H (supplemental Fig. 1). For example, structures such as Rtms5 (6Prescott 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) and KFP (5Quillin M.L. Anstrom D.M. Shu X. O'Leary S. Kallio K. Chudakov D.M. Remington S.J. Biochemistry. 2005; 44: 5774-5787Crossref PubMed Scopus (142) Google Scholar) adopt a trans non-co-planar conformation, whereas eqFP611 (7Petersen 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) has a trans co-planar conformation. Chromophore planarity can be evaluated through the dihedral angles between the imidazolinone and p-hydroxyphenyl rings (Fig. 4). Chromophores with a cis conformation, such as dsRed, have a nearly perfect co-planarity (dihedral angle <1°). In contrast, Rtms5 (6Prescott 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) and KFP (5Quillin M.L. Anstrom D.M. Shu X. O'Leary S. Kallio K. Chudakov D.M. Remington S.J. Biochemistry. 2005; 44: 5774-5787Crossref PubMed Scopus (142) Google Scholar) have a trans non-co-planar conformation with a dihedral angle of 35° and 20°, respectively. Similarly the dihedral angle of cjBlue is 40°. A second assessment of chromophore planarity is the measurement of torsion angles between χ1 (C2-Cα2-Cβ2-Cγ2) and χ2 (Cα2-Cβ2-Cγ2-Cδ2). Rtms5 (6Prescott 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) and KFP (5Quillin M.L. Anstrom D.M. Shu X. O'Leary S. Kallio K. Chudakov D.M. Remington S.J. Biochemistry. 2005; 44: 5774-5787Crossref PubMed Scopus (142) Google Scholar) have a χ1 and χ2 of 169°, -136° and 171°, -163°, respectively, whereas the torsion angles of cjBlue are 178° and -141°. Structural Comparison of cjBlue(Y64L) with Wild-type cjBlue— To examine site-specific roles of the chromophore-forming tripeptide, Gln-Tyr-Gly, we employed semi-random site-specific mutagenesis on cjBlue (19Sawano A. Miyawaki A. Nucleic Acids Res. 2000; 28: E78Crossref PubMed Scopus (303) Google Scholar). Random substitutions of these and other amino acids surrounding the chromophore were simultaneously introduced into the protein. We found that Tyr64 could be replaced by some other amino acid without losing any light absorbing ability. For example, substitution of Leu only resulted in a blue shift of the absorption peak. This variant protein, cjBlue(Y64L), was yellowish in color and absorbed light maximally at 418 nm (Fig. 2). Most FPs/CPs have Tyr at this position, whereas blue- and cyan-emitting FPs derived from Aequorea GFP have His and Trp, respectively. To investigate the structural properties of this aromatic-less chromophore, we determined the crystal structure of cjBlue(Y64L) to 2.0 Å using molecular replacement and cjBlue as the search model. A final Rfactor and Rfree of 19.8 and 24.4%, respectively, were reached. The overall quaternary and tertiary structure of cjBlue(Y64L) was found to be similar to cjBlue, but significant differences were observed in the chromophore structure and environment (Fig. 3). In comparison with the wild-type, the overall size of the cjBlue(Y64L) chromophore is smaller than that of the cjBlue chromophore. The cjBlue(Y64L) chromophore consists of a single imidazolinone ring and has the wild-type tyrosine ring replaced with an isobutyl moiety from leucine. This generates a new 5-isobutyl-imidazolinone group, which forms the cjBlue(Y64L) chromophore (Fig. 3). As compared with the cjBlue chromophore, the overall electron π-system conjugation is reduced despite the presence of an acylimine bond between the Cα and nitrogen atom of Gln63. Similar to cjBlue, the deeply buried chromophore in cjBlue(Y64L) forms extensive contacts with neighboring residues and internal water molecules (Fig. 4). The isobutyl moiety creates a new hydrogen bond network between the cjBlue(Y64L) chromophore and water molecules. Three water molecules are found in this network for chromophore stabilization within the β-barrel (Fig. 4). Water molecule W4 forms a hydrogen bond with O2 (3.2 Å) and N2 (3.5 Å) of the chromophore and a third hydrogen bond with W3 (2.4 Å), which in turn hydrogen bonds with W2 (2.8 Å) (Fig. 4). Completion of the bridging coordination between the chromophore and β-barrel is achieved by W2 hydrogen bonding to Thr158 (2.9 Å). The cjBlue(Y64L) water molecule (W1) is shifted from an equivalent water molecule (W1) position in cjBlue forming a new hydrogen bond with Glu145 (3.0 Å), Thr158 (3.0 Å), and Thr176 (2.7 Å). This point mutation also brings the chromophore into closer proximity with His197 (Fig. 5). Such proximity between His197 and the chromophore, not previously observed in cjBlue, appears to contribute to cjBlue(Y64L) absorbance. A striking difference between the cjBlue and cjBlue(Y64L) structures is that His197 assumes two different conformations at a nearly identical population within the cjBlue crystal structure. In conformation A, His197 Nϵ is 3.1 and 5.5 Å away from Cβ2 and N2 of the chromophore, respectively. In conformation B, the distance of His197 Nϵ is 4.2 and 4.6 Å away from Cβ2 and N2 of the chromophore, respectively. Conformation A, His197 has corresponding dihedral anglesχ1 of 117° andχ2 of 51°. Although His197 in conformation B has dihedral angles χ1 of -64° and χ2 of -65°. To assess the importance of His197 in the optic properties of cjBlue and cjBlue(Y64L), we generated cjBlue(H197S) and cjBlue-(Y64L/H197S) mutants. Not surprisingly, both cjBlue(H197S) and cjBlue(Y64L/H197S) yielded a colorless protein with previous absorbance peaks abolished (Fig. 2). Proper protein folding of cjBlue(H197S) and cjBlue(Y64L/H197S) was confirmed through circular dichroism analysis (data not shown). In this study, we determined the crystal structure of a blue CP, cjBlue, to 1.8 and 2.0 Å, respectively, and compared it with the crystal structures of Rtms5 (6Prescott 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) and dsRed (18Yarbrough D. Wachter R.M. Kallio K. Matz M.V. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 462-467Crossref PubMed Scopus (397) Google Scholar). We suggest that the cjBlue chromophore follows a similar formation mechanism as suggested for Rtms5 (6Prescott 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, 8Beddoe 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) and dsRed (18Yarbrough D. Wachter R.M. Kallio K. Matz M.V. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 462-467Crossref PubMed Scopus (397) Google Scholar, 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 (529) Google Scholar) (supplemental Fig. 1). However, noticeable differences are seen in the planarity of the hydroxyphenyl ring among the non-fluorescent cjBlue, the weakly fluorescent Rtms5 (6Prescott 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), and the highly fluorescent dsRed (18Yarbrough D. Wachter R.M. Kallio K. Matz M.V. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 462-467Crossref PubMed Scopus (397) Google Scholar). The cjBlue and Rtms5 chromophores adopt a trans non-co-planar conformation with the hydroxyphenyl ring rotated 40° and 35° out of plane, respectively (supplemental Fig. 2). In comparison, the dsRed chromophore adopts a cis co-planar conformation with a nearly perfect planarity (<1°). Another piece of information is that the chromophore of a highly fluorescent protein, eqFP611, has a trans co-planar conformation. Taken altogether, there is a correlation between the chromophore co-planarity and high fluorescence quantum yield. Further inspection of the cjBlue crystal structure revealed a number of residues, which contributed to the trans non-co-planar conformation of the cjBlue chromophore. First, His197 appears to stabilize chromophore conformation through parallel ring-stacking interaction with the hydroxyphenyl ring (Fig. 5). In Rtms5, Arg197 is found in the same position as His197 and favors the trans chromophore orientation through hydrogen bonding with the phenoxy group (6Prescott 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). Mutagenesis of His197 to Ser197 in cjBlue results in the loss of absorbance at 610 nm (Fig. 2), which suggests that His197 is required for catalysis of the chromophore or that the imidazole ring sustains the trans non-co-planar conformation of the chromophore. Second, interactions between the phenoxy group of the chromophore with Glu145 of β-strand 7 and Thr158 of β-strand 8 stabilize the orientation of the chromophore (Figs. 1 and 4). Of note, Rtms5 has Glu148 in β-strand 7 and Asn161 in β-strand 8 found at the equivalent position (6Prescott 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 interaction of these residues with the chromophore contributes to the chromophore conformation stability. Through semi-random mutagenesis studies, we have identified a chromophore mutant, cjBlue(Y64L) in which the hydroxyphenol group of tyrosine is replaced with the isobutyl moiety of the leucine, resulting in a significant blue shift in the absorbance spectrum (610-417 nm). Visually this mutant appears yellow in color (Fig. 2). The same blue shift was achieved by the mutagenesis of Tyr64 to Met. To investigate the structural basis for this color shift, the crystal structure of cjBlue(Y64L) was determined to 2.0 Å. The cjBlue(Y64L) chromophore structure comprises a single cyclical imidazolinone ring and forms a unique hydrogen bond network with its surrounding residues (Fig. 4); Glu145 of β-strand seven hydrogen bonds with Arg147 of β-strand 7 instead of the chromophore. Thr158 of β-strand 8 no longer interacts with the chromophore but rather forms new hydrogen bonds with other neighboring residues. Strikingly, the crystal structure of the cjBlue(Y64L) mutant revealed two alternative side-chain conformations of His197 (Fig. 5, B and C), both of which increased proximity to the chromophore relative to that in the wild-type structure. In conformation A, the distance between the side chain of His197 and the chromophore is 3.1-5.5 Å, whereas in conformation B the distance increases to 4.2-4.5 Å (Fig. 5). It is possible that these two conformations coexist in solution, thereby contributing to the blue-shifted absorbance of cjBlue(Y64L). Interestingly, the isobutyl moiety of the cjBlue(Y64L) chromophore appears to have missing electron density in our structure, suggesting that the conformational flexibility of His197 is intimately coupled with the chromophore conformation. It remains to be determined what exact contributions each conformer has to generate the yellow appearance. cjBlue and the cjBlue(Y64L) mutant set a new absorbance range (417-610 nm) for CPs. The structures of cjBlue and cjBlue(Y64L) provide valuable insights into the understanding of the non-fluorescent properties of this sea anemone protein. The cjBlue crystal structure provided a better understanding for the importance of the ring stacking effect between Tyr64 and His197. The cjBlue(Y64L) crystal structure revealed the interconversion between two His197 imidazole ring conformations on the CP optical behavior. We conclude that the dynamics and structure of the chromophore are both essential for the optical appearance of these color proteins. Future studies on the chromophore formation mechanism will further increase our knowledge of CPs and strengthen our understanding of their spectral properties. We thank the staff of 19-ID beamline at Advanced Photo Source for help on data collection and Dr. Emil Pai of the University of Toronto for his encouragement. We also thank lab members of Drs. M. Ikura and E. Pai for helpful discussions. Download .pdf (.09 MB) Help with pdf files
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