Sterically Locked Synthetic Bilin Derivatives and Phytochrome Agp1 from Agrobacterium tumefaciens Form Photoinsensitive Pr- and Pfr-like Adducts

Abstract

Phytochrome photoreceptors undergo reversible photoconversion between the red-absorbing form, Pr, and the far-red-absorbing form, Pfr. The first step in the conversion from Pr to Pfr is a Z to E isomerization around the C15=C16 double bond of the bilin chromophore. We prepared four synthetic biliverdin (BV) derivatives in which rings C and D are sterically locked by cyclizing with an additional carbon chain. In these chromophores, which are termed 15Za, 15Zs, 15Ea, and 15Es, the C15=C16 double bond is in either the Z or E configuration and the C14–C15 single bond in either the syn or anti conformation. The chromophores were assembled with Agrobacterium phytochrome Agp1, which incorporates BV as natural chromophore. All locked BV derivatives bound covalently to the protein and formed adducts with characteristic spectral properties. The 15Za adduct was spectrally similar to the Pr form and the 15Ea adduct similar to the Pfr form of the BV adduct. Thus, the chromophore of Agp1 adopts a C15=C16 Z configuration and a C14–C15 anti conformation in the Pr form and a C15=C16 E configuration and a C14–C15 anti conformation in the Pfr form. Both the 15Zs and the 15Es adducts absorbed only in the blue region of the visible spectra. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe for protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agp1. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects. Phytochrome photoreceptors undergo reversible photoconversion between the red-absorbing form, Pr, and the far-red-absorbing form, Pfr. The first step in the conversion from Pr to Pfr is a Z to E isomerization around the C15=C16 double bond of the bilin chromophore. We prepared four synthetic biliverdin (BV) derivatives in which rings C and D are sterically locked by cyclizing with an additional carbon chain. In these chromophores, which are termed 15Za, 15Zs, 15Ea, and 15Es, the C15=C16 double bond is in either the Z or E configuration and the C14–C15 single bond in either the syn or anti conformation. The chromophores were assembled with Agrobacterium phytochrome Agp1, which incorporates BV as natural chromophore. All locked BV derivatives bound covalently to the protein and formed adducts with characteristic spectral properties. The 15Za adduct was spectrally similar to the Pr form and the 15Ea adduct similar to the Pfr form of the BV adduct. Thus, the chromophore of Agp1 adopts a C15=C16 Z configuration and a C14–C15 anti conformation in the Pr form and a C15=C16 E configuration and a C14–C15 anti conformation in the Pfr form. Both the 15Zs and the 15Es adducts absorbed only in the blue region of the visible spectra. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe for protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agp1. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects. Phytochromes are photoreceptors of plants, bacteria, and fungi with a bilin chromophore that absorb light in the red and far-red region (1Lamparter T. FEBS Lett. 2004; 573: 1-5Crossref PubMed Scopus (97) Google Scholar). The natural chromophore differs between species: land plants use phytochromobilin (2Rüdiger W. Thümmler F. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis in Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 51-69Crossref Google Scholar) and cyanobacteria use phycocyanobilin (3Hübschmann T. Börner T. Hartmann E. Lamparter T. Eur. J. Biochem. 2001; 268: 2055-2063Crossref PubMed Scopus (82) Google Scholar), whereas other bacteria use biliverdin (BV) 1The abbreviations used are: BV, biliverdin; 18EtBV, 18,18′-dihydrobiliverdin; 15Za, 15Z-anti-18EtBV; 15Zs, 15Z-syn-18EtBV; 15Ea, 15E-anti-18EtBV; 15Es, 15E-syn-18EtBV; Agp1, Agrobacterium phytochrome 1; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); λmax, wavelength at absorption maximum; Pr, red-absorbing form of phytochrome; Pfr, far-red-absorbing form of phytochrome; SEC, size exclusion chromatography. 1The abbreviations used are: BV, biliverdin; 18EtBV, 18,18′-dihydrobiliverdin; 15Za, 15Z-anti-18EtBV; 15Zs, 15Z-syn-18EtBV; 15Ea, 15E-anti-18EtBV; 15Es, 15E-syn-18EtBV; Agp1, Agrobacterium phytochrome 1; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); λmax, wavelength at absorption maximum; Pr, red-absorbing form of phytochrome; Pfr, far-red-absorbing form of phytochrome; SEC, size exclusion chromatography. (4Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (245) Google Scholar). The natural chromophore attaches covalently to a cysteine residue by an autocatalytical process, although in vitro adducts with non-covalently bound chromophores may also be spectrally active (5Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar, 6Borucki B. Otto H. Rottwinkel G. Hughes J. Heyn M.P. Lamparter T. Biochemistry. 2003; 42: 13684-13697Crossref PubMed Scopus (58) Google Scholar). Agrobacterium phytochrome Agp1, which is used in the present study, belongs to the BV binding phytochromes and attaches its chromophore to Cys-20 (5Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar, 7Lamparter T. Carrascal M. Michael N. Martinez E. Rottwinkel G. Abian J. Biochemistry. 2004; 43: 3659-3669Crossref PubMed Scopus (110) Google Scholar) via the ring A vinyl side chain of BV (8Lamparter T. Michael N. Caspani O. Miyata T. Shirai K. Inomata K. J. Biol. Chem. 2003; 278: 33786-33792Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Upon assembly, the red absorption maximum (λmax) of the chromophore shifts toward a longer wavelength, accompanied by an absorbance increase, to form the red-absorbing Pr. Light absorption of Pr initiates photoconversion into the Pfr form, which results in a further ∼50–70-nm red shift of λmax. Light absorption of Pfr initiates the reverse photoconversion into Pr. For plant phytochromes it has been shown that the first step of the Pr to Pfr photoconversion is a Z to E isomerization of the chromophore around the C15=C16 double bond (9Rüdiger W. Thümmler F. Cmiel E. Schneider S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6244-6248Crossref PubMed Google Scholar), which occurs in the picosecond time scale (10Heyne K. Herbst J. Stehlik D. Esteban B. Lamparter T. Hughes J. Diller R. Biophys. J. 2002; 82: 1004-1016Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 11Holzwarth A.R. Venuti E. Braslavsky S.E. Schaffner K. Biochim. Biophys. Acta. 1992; 1140: 59-68Crossref Scopus (55) Google Scholar). Isomerization is followed by spectral changes in the microsecond and millisecond time scale (12Zhang C.F. Farrens D.L. Bjorling S.C. Song P.S. Kliger D.S. J. Amer. Chem. Soc. 1992; 114: 4569-4580Crossref Scopus (65) Google Scholar). During photoconversion, the chromophore also undergoes movements around the exocyclic single bonds. In principle, each single bond can adopt either the syn or anti conformation (13Falk H. The Chemistry of Linear Oligopyrroles and Bile Pigments. Springer Verlag Wien, New York1989Crossref Google Scholar). Vibrational spectroscopy gained indirect insight into the conformation of the phytochrome chromophore in the Pr, Pfr, and intermediate states, but the data were not unambiguous and have been interpreted in different ways (14Andel F.I. Murphy J.T. Haas J.A. McDowell M.T. van der Hoef I. Lugtenburg J. Lagarias J.C. Mathies R.A. Biochemistry. 2000; 14: 2667-2676Crossref Scopus (82) Google Scholar, 15Mizutani Y. Tokutomi S. Kitagawa T. Biochemistry. 1994; 33: 153-158Crossref PubMed Scopus (57) Google Scholar, 16Kneip C. Hildebrandt P. Schlamann W. Braslavsky S.E. Biochemistry. 1999; 16: 15185-15192Crossref Scopus (121) Google Scholar, 17Mroginski M.A. Murgida D.H. von Stetten D. Kneip C. Mark F. Hildebrandt P. J. Amer. Chem. Soc. 2004; 126: 16734-16735Crossref PubMed Scopus (77) Google Scholar). For example, it has been proposed that the formation of Pfr is accompanied by a syn/anti rotation around the C14–C15 single bond (14Andel F.I. Murphy J.T. Haas J.A. McDowell M.T. van der Hoef I. Lugtenburg J. Lagarias J.C. Mathies R.A. Biochemistry. 2000; 14: 2667-2676Crossref Scopus (82) Google Scholar). More recently, interpretation of Resonance Raman spectra by density functional theory calculations proposed that the C14–C15 single bond is in the anti conformation throughout the entire photocycle and that the C5–C6 single bond rotates from anti to syn upon conversion from Pr to Pfr (17Mroginski M.A. Murgida D.H. von Stetten D. Kneip C. Mark F. Hildebrandt P. J. Amer. Chem. Soc. 2004; 126: 16734-16735Crossref PubMed Scopus (77) Google Scholar). The Pr/Pfr photocycle is associated with changes of the protein that have been by size exclusion or J.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar, E. Song P.S. Kliger D.S. Biochemistry. PubMed Scopus Google Scholar, B. Carrascal M. Abian J. Lamparter T. Biochemistry. PubMed Scopus Google Scholar). phytochromes are histidine in which kinase activity is by changes of the activity is in the Pr and in the Pfr form, although other have also been (4Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (245) Google Scholar, T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar, S.H. Murphy J.T. Lagarias J.C. PubMed Scopus Google Scholar, T. Börner T. W. N. Eur. J. Biochem. 2001; 268: PubMed Scopus Google Scholar). changes of the protein also different of plant phytochromes Lagarias J.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T. D.S. S.H. S.H. Y. F. Song P.S. E. Full Text Full Text PDF PubMed Scopus Google Scholar) and with protein as M. Nature. 1999; PubMed Scopus Google Scholar). the of the chromophore is with the conformation of the assembled Agrobacterium Agp1 with different synthetic sterically locked bilin derivatives have been used to for the of side and to photoconversion and physiological action of plant phytochromes (8Lamparter T. Michael N. Caspani O. Miyata T. Shirai K. Inomata K. J. Biol. Chem. 2003; 278: 33786-33792Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, H. T. Inomata K. T. H. K. M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar, H. Inomata K. H. T. D. A. K. K. M. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, I. B. Braslavsky S.E. W. Schaffner K. Chem. Ed. Scopus Google Scholar). The chromophores used in the present four BV derivatives in which the rings C and D are by an additional carbon chain in either the or The adducts were analyzed by spectroscopy and protein that for the between Pr and Pfr of was from of was to the for and phycocyanobilin derivatives T. H. T. K. D. H. Inomata K. Chem. Lett. Scopus Google Scholar, T. H. Inomata K. 1999; Scopus Google Scholar, D. Inomata K. Chem. Lett. 2001; Scopus Google Scholar, S. T. H. Inomata K. Chem. Lett. 2001; Scopus Google Scholar). 15Za and 15Ea BV derivatives were prepared as H. Inomata K. Chem. Lett. Scopus Google Scholar, Y. H. Inomata K. Chem. Lett. 2004; 33: Scopus Google Scholar). of 15Zs and 15Es be of were prepared in and at and of Agrobacterium Phytochrome termed was used for Agp1. for the Agp1 protein with a for (5Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar). which for the of Agp1 followed by histidine was was by as followed by was by The protein is termed For and of Agp1 and followed the as T. Esteban B. Hughes J. Eur. J. Biochem. 2001; 268: PubMed Scopus (97) Google Scholar). In E. the were in protein was by and the were with a were by chromatography and by size exclusion chromatography The was in SEC, the was this the protein was at and spectra were as (8Lamparter T. Michael N. Caspani O. Miyata T. Shirai K. Inomata K. J. Biol. Chem. 2003; 278: 33786-33792Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) at For the was with to a of as from absorbance at and into a of or as chromophore was from the to a of the was for a was from to the of the was The first was spectra were at spectral changes were chromophores are covalently bound to the and chromophore were as and in at was to a of and the was for The was a as (5Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar, T. Michael N. Caspani O. Miyata T. Shirai K. Inomata K. J. Biol. Chem. 2003; 278: 33786-33792Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). a of absorption spectra and the of covalently bound chromophore can be In binding was also the was with 5,5′-dithiobis(2-nitrobenzoic for which the binding (5Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar). also with the that was present in the of the which has a of were was a to the of this chromophore was to the protein and a of the was a as of spectra of the was to the of the protein the chromophore which was present in the the the absorbance in the blue For photoconversion, the were for with either red light from a light or light from a light was with of chromophore and for The BV and adducts were used either or red was a at with The was with and The was at and were for was as (5Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar). The time was different chromophores were used for with Agrobacterium phytochrome biliverdin and In the four chromophores, the rings C and D are sterically locked by an additional carbon chain in of four the C15=C16 double bond is in either the Z or E configuration and the single bond in either the syn or anti conformation the natural chromophore of Agp1 (5Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar), and as Both chromophores form a bond with the and adducts that reversible photoconversion (8Lamparter T. Michael N. Caspani O. Miyata T. Shirai K. Inomata K. J. Biol. Chem. 2003; 278: 33786-33792Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Phytochrome chromophore can be followed by Phytochrome chromophores have absorption in the blue and red Upon into the chromophore the red shifts to longer and the absorbance the of the blue absorption The BV adduct of Agp1 has the as the but the of the is (5Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar, T. Michael N. Caspani O. Miyata T. Shirai K. Inomata K. J. Biol. Chem. 2003; 278: 33786-33792Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). the from the to the is by an absorbance In present study, the absorbance was between and chromophore and protein The Pr in the red spectral region was at with was also between and and the Pr adduct a red at The absorption maximum is with that of the of is that of BV by the of a vinyl at the 15Za, a similar was as with BV and The of the adduct has a form with absorption in the red and the and a red at The absorption of this adduct are with the Pr form of the BV and the absorption in the red and the blue phytochromes have the absorption in the red spectral which at the red absorption maximum and as a in the spectra of the BV and adducts a and In the 15Za this as the with a at spectral between the adducts is the absorbance between the red and blue absorption which from BV to 15Za of BV 15Za 15Zs 15Ea 15Es in and chromophore with Agp1. and chromophore were and spectra and in the The of was in the of the was The chromophore was in the of BV in the of and in other spectra were to of The spectra of chromophores were spectral of Agp1 chromophore Pr or in blue spectral of side in red spectral in red spectral red is only the not be is only the not be in a Both 15Zs and 15Es chromophores spectrally chromophore and the red absorption was and data that the between chromophore and protein is and The adducts have only a blue absorption with a at for 15Zs and for protein and 15Ea an absorption with a at and to to spectral changes were the first differs from the of the the is also The spectral changes an the formation of the The of the adduct is similar to the Pfr spectra of the BV the adducts a and which have a at and The of the red and blue absorption of and 15Ea adducts are and The of the side is also and the absorbance are similar has been shown that the first step in the Pr to Pfr photoconversion of plant phytochromes is a Z to E isomerization around the C15=C16 double bond (9Rüdiger W. Thümmler F. Cmiel E. Schneider S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6244-6248Crossref PubMed Google Scholar). of the spectral this is for although is Agp1 the adducts with locked chromophores in the not spectral changes upon with either red or light that of the BV and adducts a and and of the the chromophores are covalently bound to the chromophore and protein and was to and the were to In this way, the protein is separated from chromophore. spectra and this the of covalently bound chromophore can be (5Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar, T. Michael N. Caspani O. Miyata T. Shirai K. Inomata K. J. Biol. Chem. 2003; 278: 33786-33792Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). that chromophores bound in a to the Agp1 protein were with BV and 15Ea in which the time was only these BV was bound not the of bound 15Ea was is in with the that the spectral changes 15Ea the formation of the We a similar the chromophore binding by In this case, was in the was spectra and In this way, the of the was the of the In the chromophores bound to the protein the Thus, chromophores with the The of to is the and of the In with phytochrome between Pr, Pfr, and the T. Esteban B. Hughes J. Eur. J. Biochem. 2001; 268: PubMed Scopus (97) Google Scholar). Pr/Pfr were for of that the histidine kinase B. Carrascal M. Abian J. Lamparter T. Biochemistry. PubMed Scopus Google Scholar, H. Lamparter T. B. Hughes J. Heyn M.P. Biochemistry. 2003; 42: PubMed Scopus Google Scholar). We Agp1 as as Pr and Pfr of the BV adduct to but between these We also and the chromophore termed which from to and the histidine kinase The of and the Pr and Pfr of the BV adduct of were these to of and The results with Pr and Pfr of the adduct were with of the BV adduct The 15Za adduct with an of in between the Pr of and the The of the 15Zs and 15Es adducts were to that of the and whereas the of the 15Ea adduct was with that of the Pfr form of the BV and adducts the of the is that in the protein formed and that the in time from changes of the of the histidine kinase activity of Agp1 is light upon photoconversion from Pr to Pfr, the activity of Agp1 We of different Agp1 adducts to for the of the locked chromophores kinase activity In these the activity of was to The activity of the was The activity of the as with the Pr form of the adduct is in with (5Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar), although in present the was not of different of Upon conversion into Pfr, the activity was to in with (5Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar). The data of the adduct were with of the BV the activity of Pr was and that of Pfr The activity of the 15Za adduct was and thus with that of the but between 15Za and the Pr of the BV and adducts were not The activity of the 15Ea adduct was only and thus with the activity of Pfr of BV and adducts of Agp1. be that 15Ea holoprotein adopt the whereas in the of the red BV and only Pfr were The data of the 15Ea adduct that Pfr has a a be from as red of natural chromophore The 15Zs and 15Es adducts and data that the syn chromophores a conformation the protein that is different from the the Pr, and the Pfr form. In the present study, four different synthetic BV chromophores in which rings C and D are locked were for with Agrobacterium phytochrome Agp1. with spectral that chromophores with the Agp1 protein and form A of the absorption spectra and the conformation and configuration of the natural chromophore the Agp1 chromophore The 15Za adduct is spectrally similar to the Pr form of or and that in Pr the chromophore adopts a C15=C16 Z configuration and C14–C15 anti conformation. In plant the configuration of the double which is protein is from (9Rüdiger W. Thümmler F. Cmiel E. Schneider S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6244-6248Crossref PubMed Google Scholar). anti conformation of the single bond the E configuration of the double has been proposed from spectroscopy C. Hildebrandt P. Schlamann W. Braslavsky S.E. Biochemistry. 1999; 16: 15185-15192Crossref Scopus (121) Google Scholar), but a C14–C15 syn conformation has also been S. T. M. 1992; Scopus Google Scholar). The locked chromophore that an adduct similar to Pfr is in the Pfr form the chromophore has a C15=C16 E configuration and a C14–C15 anti conformation. The C15=C16 E configuration of plant phytochrome Pfr has also been shown by (9Rüdiger W. Thümmler F. Cmiel E. Schneider S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6244-6248Crossref PubMed Google Scholar). a C14–C15 syn conformation has been proposed for the Pfr form C. Hildebrandt P. Schlamann W. Braslavsky S.E. Biochemistry. 1999; 16: 15185-15192Crossref Scopus (121) Google Scholar). density functional theory calculations that the Pfr chromophore is in the C14–C15 anti conformation (17Mroginski M.A. Murgida D.H. von Stetten D. Kneip C. Mark F. Hildebrandt P. J. Amer. Chem. Soc. 2004; 126: 16734-16735Crossref PubMed Scopus (77) Google Scholar), in with present The of 15Ea is different from the other chromophores it longer the absorbance changes are The which is formed has a far-red absorbance and a at The adduct has a absorbance and a at The Pfr form of and adducts or binding have a absorbance and a as with the Pfr form of the adducts (5Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar, 6Borucki B. Otto H. Rottwinkel G. Hughes J. Heyn M.P. Lamparter T. Biochemistry. 2003; 42: 13684-13697Crossref PubMed Scopus (58) Google Scholar). We thus proposed that the from the to the adduct the formation of the was by the results are in with a in which Agp1 was assembled with BV T. Michael N. Scholar). In this study, a with absorbance and a was The chromophore was not covalently bound to the bond formation was by into is the bond with 15Ea formed as with the other be that the of 15Ea into the Agp1 chromophore the between the Cys-20 and the ring A vinyl side chain of 15Ea is and that the for bond formation is In this of the protein the chromophore be for bond For the an rotation around the C5–C6 single bond is the in vitro phytochrome adducts can undergo photoconversion (5Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (161) Google Scholar, 6Borucki B. Otto H. Rottwinkel G. Hughes J. Heyn M.P. Lamparter T. Biochemistry. 2003; 42: 13684-13697Crossref PubMed Scopus (58) Google Scholar), phytochrome have a covalently bound chromophore in The present results that bond formation is for Pfr with spectral properties. is the of the locked chromophores the conformation of the Agp1 this have and and The activity of Pfr is that of Pr. For the chromophore was the of adducts is in the Pfr in the Pr form. The was also in but between Pr and the were and the not to between The 15Za adducts like Pr of the BV and adducts and the In which between and the Pr form, the of the 15Za adduct was from that of the Pr form not For this that the protein conformation of the 15Za adduct is similar to or with the Pr form of Agp1. The 15Ea adducts are similar to the Pfr of the BV and adducts in and data that a form of phytochrome can be in darkness. the E is the protein adopts its conformation in a ways for phytochrome 15Ea chromophores can be to plants that are in the of the phytochrome chromophore. The chromophores be by the plants, as shown for other natural and synthetic chromophores H. T. Inomata K. T. H. K. M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar, PubMed Google Scholar, T. H. D. Hughes J. Hartmann E. Scopus Google Scholar). the plant phytochrome with the locked 15Ea a form be of the light In this way, Pfr can be from other light and can be separated from of Pr/Pfr The results with the 15Zs and 15Es chromophores that the Agp1 protein is for that have the conformation. The absorption spectra of adducts are similar to of adducts as to phytochromes or other A absorption with a blue absorption at was with the bilin binding of S.H. Lagarias J.C. Biochemistry. 2000; PubMed Scopus Google Scholar). Both locked syn adopt a conformation the Agp1 chromophore as with the conformation of other be that the of chromophore is by a of an side chain to the the be the red absorption upon of the as for Both and adducts behaved like the but an the protein can adopt at four different with to its kinase the form of the Pr, Pfr, and that of the 15Zs or 15Es adduct. is the kinase of phytochromes is by the chromophore to a B. Carrascal M. Abian J. Lamparter T. Biochemistry. PubMed Scopus Google Scholar), the and C of the phytochrome protein be as of a a of In this Pr to Pfr photoconversion the between kinase the between chromophore is upon Pr/Pfr of locked syn chromophores into Agp1 in an of the between chromophore and in this have a kinase