Structural Characterization of a Blue Chromoprotein and Its Yellow Mutant from the Sea Anemone Cnidopus Japonicus

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. 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 The 5′ and 3′ of the cDNA were amplified using the amplification of cDNA The amino with sequence 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), S.J. Kallio K. Lukyanov K.A. Biochemistry. 2005; 44: PubMed Scopus Google Scholar), 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) The full-length protein was expressed in coli with a His6 tag at the and purified using The protein referred as cjBlue is a which not fluoresce but is green in at a maximum at 610 nm = the of CPs. The spectrum of cjBlue to to not of cjBlue and mutant of a temperature of 22 °C, and the of CPs were used to the absorbance cjBlue is in cjBlue(Y64L) in in and cjBlue(Y64L/H197S) in Structure of crystal structure of cjBlue, solved by was to 1.8 Å. The cjBlue crystal belongs to the P21 space and of and were The cjBlue model contained and in an unit. the structure of cjBlue a similar fold to GFP M. Kallio K. R.Y. Remington S.J. PubMed Scopus Google Scholar), dsRed M.A. M. R. Nat. Biol. 2000; 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). of a with a with the of the The of cjBlue the chromophore to the of the protein and for cjBlue and = × is the of for and is the of the of = 100 × and are the and structure as for for of the not used for model = × is the of for and is the of the = 100 × and are the and structure as for for of the not used for model in a new The crystal structure of cjBlue revealed a of four that were in with in a similar to that for 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 M.A. M. R. Nat. Biol. 2000; PubMed Scopus (302) Google Scholar). This molecular is stabilized by and of the with of of and are at the between The interaction between and at and and of an of The cjBlue chromophore of a group with an bond found between the and nitrogen at and (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 bond at is with the bond found at of dsRed M.A. M. R. Nat. Biol. 2000; PubMed Scopus (302) Google Scholar, Kallio K. Matz M.V. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). FPs and the cjBlue chromophore is the with to and internal and which are to the of the ring near the cjBlue chromophore were found in the same as 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 near the a stacking with the ring of Å. The cjBlue chromophore a trans conformation between the Cα2-Cβ2 bond with bond of internal between and an in the bond and the bond was also to to the trans conformation by the ring from a cis a trans with the exception of eqFP611, between the and ring of internal of and 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) a trans 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 can be through the between the and with a cis such as have a co-planarity In 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 conformation with a of and the of cjBlue is of chromophore is the of between 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) 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 and of and the of cjBlue are and of cjBlue(Y64L) with To of the tripeptide, we employed semi-random on cjBlue (19Sawano A. Miyawaki A. Nucleic Acids Res. 2000; 28: E78Crossref PubMed Scopus (303) Google Scholar). of these and amino the chromophore were introduced into the protein. We found that Tyr64 be by amino acid of Leu only in a blue shift of the This variant cjBlue(Y64L), was in color and at nm have at this and FPs from Aequorea GFP have and To the structural properties of this we the crystal structure of cjBlue(Y64L) to 2.0 Å using molecular replacement and cjBlue as the search and of and were reached. The and structure of cjBlue(Y64L) was found to be similar to cjBlue, but were in the chromophore structure and environment In with the the size of the cjBlue(Y64L) chromophore is than that of the cjBlue chromophore. The cjBlue(Y64L) chromophore of a single ring and has the wild-type ring with an from This a new which the cjBlue(Y64L) chromophore with the cjBlue the is the of an bond between the and nitrogen of to cjBlue, the chromophore in cjBlue(Y64L) with and internal The a new bond between the cjBlue(Y64L) chromophore and are found in this for chromophore the a bond with and of the chromophore and a bond with which in with of the between the chromophore and is by to The cjBlue(Y64L) is from an position in cjBlue a new bond with and This mutation also the chromophore into with His197 between His197 and the not previously in cjBlue, to to cjBlue(Y64L) between the cjBlue and cjBlue(Y64L) structures is that His197 two different conformations at a the cjBlue crystal In conformation His197 is and Å from and of the In conformation the of His197 is and Å from and of the His197 has of of His197 in conformation has of and of To the of His197 in the properties of cjBlue and cjBlue(Y64L), we generated and both and cjBlue(Y64L/H197S) a protein with absorbance protein of and cjBlue(Y64L/H197S) was confirmed through not In this we the crystal structure of a blue CP, cjBlue, to 1.8 and 2.0 Å, and 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 Kallio K. Matz M.V. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). We that the cjBlue chromophore a similar formation as 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, 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 Kallio K. Matz M.V. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R.Y. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar) are in the of the ring among the non-fluorescent cjBlue, the 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 fluorescent dsRed Kallio K. Matz M.V. Remington S.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The cjBlue and Rtms5 chromophores a trans conformation with the ring and of In the dsRed chromophore a cis co-planar conformation with a of is that the chromophore of a fluorescent eqFP611, has a trans co-planar is a between the chromophore co-planarity and of the cjBlue crystal structure revealed a of which to the trans conformation of the cjBlue chromophore. His197 to chromophore conformation through interaction with the ring In is found in the same position as His197 and the trans chromophore through with the 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). of His197 to in cjBlue in the of absorbance at 610 nm which that His197 is for of the chromophore or that the imidazole ring the trans conformation of the chromophore. between the group of the chromophore with of and of 8 the of the chromophore 1 and Rtms5 has in and in 8 found at the 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 with the chromophore contributes to the chromophore conformation semi-random we have identified a chromophore mutant, cjBlue(Y64L) in which the group of is with the of the in a blue shift in the absorbance spectrum this mutant yellow in color The same blue shift was by the of Tyr64 to To the structural basis for this color the crystal structure of cjBlue(Y64L) was to 2.0 Å. The cjBlue(Y64L) chromophore structure a single ring and a unique bond with its of with of of the chromophore. of 8 no with the chromophore but new with the crystal structure of the cjBlue(Y64L) mutant revealed two conformations of His197 and both of which to the chromophore to that in the wild-type In conformation the between the of His197 and the chromophore is Å, in conformation the to Å is that these two conformations in to the absorbance of Interestingly, the of the cjBlue(Y64L) chromophore to have in our suggesting that the of His197 is with the chromophore to be each has to the yellow cjBlue and the cjBlue(Y64L) mutant a new absorbance range for CPs. The structures of cjBlue and cjBlue(Y64L) into the understanding of the non-fluorescent properties of this sea anemone protein. The cjBlue crystal structure a better understanding for the of the ring stacking between Tyr64 and The cjBlue(Y64L) crystal structure revealed the interconversion between two His197 imidazole ring conformations on the CP optical We conclude that the dynamics and structure of the chromophore are both essential for the optical appearance of these color proteins. studies on the chromophore formation our of CPs and our understanding of their spectral We the of 19-ID beamline at Advanced Source for on data and of the of for We also members of M. and for with

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.