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• W2069893600 abstract "The ability of phytochromes (Phy) to act as photointerconvertible light switches in plants and microorganisms depends on key interactions between the bilin chromophore and the apoprotein that promote bilin attachment and photointerconversion between the spectrally distinct red light-absorbing Pr conformer and far red light-absorbing Pfr conformer. Using structurally guided site-directed mutagenesis combined with several spectroscopic methods, we examined the roles of conserved amino acids within the bilin-binding domain of Deinococcus radiodurans bacteriophytochrome with respect to chromophore ligation and Pr/Pfr photoconversion. Incorporation of biliverdin IXα (BV), its structure in the Pr state, and its ability to photoisomerize to the first photocycle intermediate are insensitive to most single mutations, implying that these properties are robust with respect to small structural/electrostatic alterations in the binding pocket. In contrast, photoconversion to Pfr is highly sensitive to the chromophore environment. Many of the variants form spectrally bleached Meta-type intermediates in red light that do not relax to Pfr. Particularly important are Asp-207 and His-260, which are invariant within the Phy superfamily and participate in a unique hydrogen bond matrix involving the A, B, and C pyrrole ring nitrogens of BV and their associated pyrrole water. Resonance Raman spectroscopy demonstrates that substitutions of these residues disrupt the Pr to Pfr protonation cycle of BV with the chromophore locked in a deprotonated Meta-Rc-like photoconversion intermediate after red light irradiation. Collectively, the data show that a number of contacts contribute to the unique photochromicity of Phy-type photoreceptors. These include residues that fix the bilin in the pocket, coordinate the pyrrole water, and possibly promote the proton exchange cycle during photoconversion. The ability of phytochromes (Phy) to act as photointerconvertible light switches in plants and microorganisms depends on key interactions between the bilin chromophore and the apoprotein that promote bilin attachment and photointerconversion between the spectrally distinct red light-absorbing Pr conformer and far red light-absorbing Pfr conformer. Using structurally guided site-directed mutagenesis combined with several spectroscopic methods, we examined the roles of conserved amino acids within the bilin-binding domain of Deinococcus radiodurans bacteriophytochrome with respect to chromophore ligation and Pr/Pfr photoconversion. Incorporation of biliverdin IXα (BV), its structure in the Pr state, and its ability to photoisomerize to the first photocycle intermediate are insensitive to most single mutations, implying that these properties are robust with respect to small structural/electrostatic alterations in the binding pocket. In contrast, photoconversion to Pfr is highly sensitive to the chromophore environment. Many of the variants form spectrally bleached Meta-type intermediates in red light that do not relax to Pfr. Particularly important are Asp-207 and His-260, which are invariant within the Phy superfamily and participate in a unique hydrogen bond matrix involving the A, B, and C pyrrole ring nitrogens of BV and their associated pyrrole water. Resonance Raman spectroscopy demonstrates that substitutions of these residues disrupt the Pr to Pfr protonation cycle of BV with the chromophore locked in a deprotonated Meta-Rc-like photoconversion intermediate after red light irradiation. Collectively, the data show that a number of contacts contribute to the unique photochromicity of Phy-type photoreceptors. These include residues that fix the bilin in the pocket, coordinate the pyrrole water, and possibly promote the proton exchange cycle during photoconversion. The phytochrome (Phy) 5The abbreviations used are: Phy, phytochrome; BphP, bacteriophytochrome; BV, biliverdin IXα; CBD, chromophore-binding domain; GAF, cGMP phosphodiesterase/adenylcyclase/FhlA; HO, heme oxygenase; ip, in-plane bending; PAS, Per/Arndt/Sim; PCB, phycocyanobilin; PΦB, phytochromobilin; PHY, phytochrome domain; PPIXa, protoporphyrin IXa; Pr, red light-absorbing state of phytochrome; Pfr, far red light-absorbing state of phytochrome; RR, resonance Raman. 5The abbreviations used are: Phy, phytochrome; BphP, bacteriophytochrome; BV, biliverdin IXα; CBD, chromophore-binding domain; GAF, cGMP phosphodiesterase/adenylcyclase/FhlA; HO, heme oxygenase; ip, in-plane bending; PAS, Per/Arndt/Sim; PCB, phycocyanobilin; PΦB, phytochromobilin; PHY, phytochrome domain; PPIXa, protoporphyrin IXa; Pr, red light-absorbing state of phytochrome; Pfr, far red light-absorbing state of phytochrome; RR, resonance Raman. superfamily encompasses a large and diverse set of photoreceptors present in the plant, fungal, and bacterial kingdoms where they play critical roles in various light-regulated processes (1Rockwell N.C. Su Y.S. Lagarias J.C. Annu. Rev. Plant Biol. 2006; 57: 837-858Crossref PubMed Scopus (780) Google Scholar, 2Vierstra R.D., and Karniol, B. (2005) in Handbook of Photosensory Receptors (Briggs, W. R., and Spudich, J. L., eds) pp. 171-196, Wiley-VCH Press, Weinheim, GermanyGoogle Scholar, 3Quail P.H. Nat. Rev. Mol. Cell Biol. 2002; 3: 85-93Crossref PubMed Scopus (559) Google Scholar). These processes range from the control of phototaxis, pigmentation, and photosynthetic potential in proteobacteria and cyanobacteria to seed germination, chloroplast development, shade avoidance, and flowering time in higher plants. Phys are unique among photoreceptors in being able to assume two stable, photointerconvertible conformers, designated Pr and Pfr based on their respective absorption maxima in the red and far-red spectral regions. By cycling between Pr and Pfr, Phys act as light-regulated switches in various photosensory cascades. Phys are homodimeric complexes with each polypeptide containing a single bilin (or linear tetrapyrrole) chromophore, which binds autocatalytically via a thioether linkage to a positionally conserved cysteine (1Rockwell N.C. Su Y.S. Lagarias J.C. Annu. Rev. Plant Biol. 2006; 57: 837-858Crossref PubMed Scopus (780) Google Scholar, 2Vierstra R.D., and Karniol, B. (2005) in Handbook of Photosensory Receptors (Briggs, W. R., and Spudich, J. L., eds) pp. 171-196, Wiley-VCH Press, Weinheim, GermanyGoogle Scholar, 3Quail P.H. Nat. Rev. Mol. Cell Biol. 2002; 3: 85-93Crossref PubMed Scopus (559) Google Scholar). The photosensing portion typically contains Per/Arndt/Sim (PAS) and cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains, which are essential for bilin binding and Pr assembly, and together comprise the chromophore-binding domain (CBD). The CBD is often followed by a Phytochrome (PHY) domain, which is required for the formation and stability of Pfr. C-terminal to the PHY domain can be a variety of domains that promote signal transmission and/or dimerization. Often histidine kinase or histidine kinase-related domains are present that can direct light-modulated phosphorylation in cis within the homodimer and in trans to other proteins, which typically are response regulator proteins in microorganisms (1Rockwell N.C. Su Y.S. Lagarias J.C. Annu. Rev. Plant Biol. 2006; 57: 837-858Crossref PubMed Scopus (780) Google Scholar, 2Vierstra R.D., and Karniol, B. (2005) in Handbook of Photosensory Receptors (Briggs, W. R., and Spudich, J. L., eds) pp. 171-196, Wiley-VCH Press, Weinheim, GermanyGoogle Scholar, 4Yeh K.C. Lagarias J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13976-13981Crossref PubMed Scopus (351) Google Scholar, 5Karniol B. Wagner J.R. Walker J.M. Vierstra R.D. Biochem. J. 2005; 392: 103-116Crossref PubMed Scopus (170) Google Scholar). Biliverdin IXα (BV) is the native chromophore for the proteobacterial and fungal Phys; it is synthesized by oxidative cleavage of heme by a heme oxygenase (HO) (6Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (245) Google Scholar, 7Froehlich A.C. Noh B. Vierstra R.D. Loros J. Dunlap J.C. Eukaryot. Cell. 2005; 4: 2140-2152Crossref PubMed Scopus (124) Google Scholar). BV is then attached to a cysteine residue upstream of the PAS domain via the C32 carbon of the pyrrole ring A vinyl side chain (8Wagner J.R. Zhang J. Brunzelle J.S. Vierstra R.D. Forest K.T. J. Biol. Chem. 2007; 282: 12298-12309Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 9Lamparter T. Carrascal M. Michael N. Martinez E. Rottwinkel G. Abian J. Biochemistry. 2004; 43: 3659-3669Crossref PubMed Scopus (110) Google Scholar). In contrast, cyanobacterial and higher plant Phys incorporate phycocyanobilin (PCB) and phytochromobilin (PΦB), respectively, which are generated from BV, in part, by enzymatic reduction of the C3 vinyl side chain on pyrrole ring A to generate an ethylidene group (1Rockwell N.C. Su Y.S. Lagarias J.C. Annu. Rev. Plant Biol. 2006; 57: 837-858Crossref PubMed Scopus (780) Google Scholar, 10Lagarias J.C. Rapoport H. J. Amer. Chem. Soc. 1980; 102: 4821-4828Crossref Scopus (258) Google Scholar). PCB and PΦB then bind via the C31 carbon of this side chain to a cysteine within the GAF domain, which is predicted to extend toward the same space as the N-terminal binding-site cysteine used by proteobacterial/fungal Phys (8Wagner J.R. Zhang J. Brunzelle J.S. Vierstra R.D. Forest K.T. J. Biol. Chem. 2007; 282: 12298-12309Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 11Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (427) Google Scholar). Despite intensive study, the unique photochromic nature of Phys remains largely unexplained. Thus far, most of our understanding has been derived from absorption and resonance Raman (RR) spectroscopy, which have collectively identified several distinct but ill-defined steps during Pr → Pfr photoconversion (Fig. 1A). As Pr, the bilin is cationic with all four of the pyrrole ring nitrogens protonated (12Kneip C. Hildebrandt P. Schlamann W. Braslavsky S.E. Mark F. Schaffner K. Biochemistry. 1999; 38: 15185-15192Crossref PubMed Scopus (121) Google Scholar, 13von Stetten D. Seibeck S. Michael N. Scheerer P. Mroginski M.A. Murgida D.H. Krauss N. Heyn M.P. Hildebrandt P. Borucki B. Lamparter T. J. Biol. Chem. 2007; 282: 2116-2123Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Using plant Phys as the example, photoexcitation with red light converts Pr (absorption maximum of the Q band at 666 nm) within picoseconds to the partially bleached Lumi-R photoproduct (absorption maximum at 688 nm) (14Andel F. Hasson K.C. Gai F. Anfinrud P.A. Mathies R.A. Biospect. 1997; 3: 421-433Crossref Scopus (46) Google Scholar, 15Bischoff M. Hermann G. Rentsch S. Strehlow D. Biochemistry. 2001; 40: 181-186Crossref PubMed Scopus (49) Google Scholar). This conversion is predicted to involve a Z to E isomerization of the C15–C16 methine double bond between the C and D pyrrole rings of the chromophore (16Andel 3rd, F. Lagarias J.C. Mathies R.A. Biochemistry. 1996; 35: 15997-16008Crossref PubMed Scopus (100) Google Scholar, 17Mizutani Y. Tokutomi S. Kitagawa T. Biochemistry. 1994; 33: 153-158Crossref PubMed Scopus (57) Google Scholar, 18Rudiger W. Thummler F. Cmiel E. Schneider S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6244-6248Crossref PubMed Google Scholar). On the microsecond time scale, thermal relaxation steps lead to the formation of the Meta-Ra intermediate with a maximum absorption at 663 nm (19Tu S.-L., and Lagarias, J. C. (2005) in Handbook of Photosensory Receptors (Briggs, W. R., and Spudich, J. L., eds) pp. 121-149, Wiley-VCH Press, Weinheim, GermanyGoogle Scholar). The Meta-Ra intermediate in turn decays in microto milliseconds to a deprotonated Meta-Rc intermediate with an absorption maximum at 725 nm (20Foerstendorf H. Benda C. Gartner W. Storf M. Scheer H. Siebert F. Biochemistry. 2001; 40: 14952-14959Crossref PubMed Scopus (73) Google Scholar, 21Braslavsky, S. E. (2003) in Photochromisms, Molecules and Systems (BrouasLaurent, D. H., and BrouasLaurent, H., eds) pp. 738-755, Elsevier Science BV, AmsterdamGoogle Scholar). Finally, the Meta-Rc photoproduct decays to Pfr (absorption maximum at 730 nm) on a millisecond time scale in a process that likely requires reprotonation of the chromophore (12Kneip C. Hildebrandt P. Schlamann W. Braslavsky S.E. Mark F. Schaffner K. Biochemistry. 1999; 38: 15185-15192Crossref PubMed Scopus (121) Google Scholar, 16Andel 3rd, F. Lagarias J.C. Mathies R.A. Biochemistry. 1996; 35: 15997-16008Crossref PubMed Scopus (100) Google Scholar, 17Mizutani Y. Tokutomi S. Kitagawa T. Biochemistry. 1994; 33: 153-158Crossref PubMed Scopus (57) Google Scholar, 22Borucki B. von Stetten D. Seibeck S. Lamparter T. Michael N. Mroginski M.A. Otto H. Murgida D.H. Heyn M.P. Hildebrandt P. J. Biol. Chem. 2005; 280: 34358-34364Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 23Andel 3rd, F. Murphy J.T. Haas J.A. McDowell M.T. van der Hoef I. Lugtenburg J. Lagarias J.C. Mathies R.A. Biochemistry. 2000; 39: 2667-2676Crossref PubMed Scopus (82) Google Scholar, 24Mroginski M.A. Murgida D.H. von Stetten D. Kneip C. Mark F. Hildebrandt P. J. Am. Chem. Soc. 2004; 126: 16734-16735Crossref PubMed Scopus (77) Google Scholar, 25Kneip C. Schlamann W. Braslavsky S.E. Hildebrandt P. Schaffner K. FEBS Lett. 2000; 482: 252-256Crossref PubMed Scopus (11) Google Scholar). Coupled with these chromophore relaxation steps are conformational changes within the polypeptide that alter the structure of Pfr versus Pr, which in turn are presumed to affect the kinase activity of the photoreceptor and/or its photoreversible interaction(s) with downstream signaling partner(s) (1Rockwell N.C. Su Y.S. Lagarias J.C. Annu. Rev. Plant Biol. 2006; 57: 837-858Crossref PubMed Scopus (780) Google Scholar, 2Vierstra R.D., and Karniol, B. (2005) in Handbook of Photosensory Receptors (Briggs, W. R., and Spudich, J. L., eds) pp. 171-196, Wiley-VCH Press, Weinheim, GermanyGoogle Scholar, 3Quail P.H. Nat. Rev. Mol. Cell Biol. 2002; 3: 85-93Crossref PubMed Scopus (559) Google Scholar). Once formed, Pfr will slowly revert nonphotochemically back to Pr or can be photoconverted rapidly back to Pr with far-red light (Fig. 1A). Neither pathway is well understood. The photoinduced Pfr → Pr conversion likely proceeds via a pathway distinct from that for the Pr → Pfr conversion but may use a similar but inverted proton migration cycle (1Rockwell N.C. Su Y.S. Lagarias J.C. Annu. Rev. Plant Biol. 2006; 57: 837-858Crossref PubMed Scopus (780) Google Scholar, 21Braslavsky, S. E. (2003) in Photochromisms, Molecules and Systems (BrouasLaurent, D. H., and BrouasLaurent, H., eds) pp. 738-755, Elsevier Science BV, AmsterdamGoogle Scholar). To help understand the photochromicity of Phys at the atomic level, the three-dimensional structures of the Pr and Pfr forms would clearly be helpful. Recently, we accomplished part of this objective by determining the structure of the CBD as Pr, using the sole bacteriophytochrome photoreceptor (BphP) in Deinococcus radiodurans assembled with BV as the source. The original structure solved to 2.5 Å resolution (11Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (427) Google Scholar), followed by a more refined model at 1.45 Å resolution (8Wagner J.R. Zhang J. Brunzelle J.S. Vierstra R.D. Forest K.T. J. Biol. Chem. 2007; 282: 12298-12309Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), revealed that the GAF domain forms a deep pocket that cradles the Pr chromophore in a ZZZsyn,syn,anti configuration corresponding to a Z geometry for all methine bridge double bonds and a syn geometry for the A–B and B–C and an anti geometry from the C–D methine bridge single bonds (Fig. 1B). Other than positioning Cys-24 that forms the thioether linkage, the PAS domain has no direct contact with BV in the Pr conformer. Instead, it is connected indirectly to the bilin and the GAF domain through a rare figure-of-eight knot in the polypeptide (Fig. 1C). The structures also identified a heretofore unknown dimerization interface between sister GAF domains that could impact signaling within the Phy dimer and/or photochemical cooperatively between the adjacent bilins (8Wagner J.R. Zhang J. Brunzelle J.S. Vierstra R.D. Forest K.T. J. Biol. Chem. 2007; 282: 12298-12309Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). A similar crystallographic model was recently described by Yang et al. (26Yang X. Stojkovic E.M. Kuk J. Moffat K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 12571-12576Crossref PubMed Scopus (158) Google Scholar) for BphP3 from the photosynthetic bacterium Rhodopseudomonas palustris, suggesting that the CBDs of all Phys have related tertiary structures. Inspection of the bilin pocket of the D. radiodurans and R. palustris BphP CBDs revealed a number of amino acids that could be important for covalent binding of the bilin, the unique spectral properties of Pr and Pfr, and the steps required for their interconversion (8Wagner J.R. Zhang J. Brunzelle J.S. Vierstra R.D. Forest K.T. J. Biol. Chem. 2007; 282: 12298-12309Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 11Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (427) Google Scholar, 26Yang X. Stojkovic E.M. Kuk J. Moffat K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 12571-12576Crossref PubMed Scopus (158) Google Scholar) (Fig. 1, B and C). Most of these amino acids are highly conserved throughout the Phy superfamily, further supporting their functional significance (5Karniol B. Wagner J.R. Walker J.M. Vierstra R.D. Biochem. J. 2005; 392: 103-116Crossref PubMed Scopus (170) Google Scholar, 11Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (427) Google Scholar). Here we tested the importance of many of these residues through the analysis of engineered protein variants by absorption, fluorescence, and RR spectroscopy. Surprisingly, although nearly all of the DrBphP variants retained their ability to fold correctly, covalently attach BV, and form Pr, a majority failed to properly photoconvert to Pfr. In particular, we demonstrated the importance to Pr → Pfr photoconversion of contacts involving the propionate side chains of BV, the hydrophobic pocket that surrounds the D pyrrole ring, and the highly conserved Asp-207 and His-260 residues, which participate in an extensive hydrogen bond network involving several ordered waters and the bilin. Even subtle substitutions of Asp-207 became trapped in a deprotonated and presumably Meta-Rc-like state after red light irradiation. Collectively, the mutant analysis revealed that although assembly of the Pr form and photoconversion to the Meta-R intermediates are generally insensitive to most changes in the chromophore pocket, complete photoconversion to the Pfr form is strongly compromised, suggesting that the transition from the Meta-R intermediates to Pfr is a dynamic process requiring a number of critical protein/protein and protein/chromophore contacts. Site-directed Mutagenesis and Protein Purification—The full-length D. radiodurans BphP (encoding 755 amino acids) gene (27Davis S.J. Vener A.V. Vierstra R.D. Science. 1999; 286: 2517-2520Crossref PubMed Scopus (283) Google Scholar) was PCR-amplified from genomic DNA using primers designed to introduce BamHI and XhoI sites before and after the designated length of coding region, respectively. The BamHI-XhoI-digested PCR products were cloned into pET21b(+) (Novagen, Madison, WI), which was similarly digested, resulting in the addition of codons for an N-terminal T7 tag and codons for a His6 tag just before the stop codon. All site-directed mutations were introduced by the PCR-based QuickChange method (Stratagene, La Jolla, CA). Each coding region was sequenced completely by the dideoxy method to confirm introduction of the appropriate mutation(s) and the absence of secondary mutations. The DrBphP variants were expressed in Escherichia coli strain Rosetta (DE3) (Novagen, Madison WI) either by themselves or simultaneously with the Synechocystis PCC6803 HO gene cloned into pET 24a(+) (Novagen) (6Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (245) Google Scholar). After harvesting the cells, all further steps were performed under green safe lights. Cells were homogenized in 30 mm Tris-HCl (pH 8.0), 200 mm NaCl, and 5 mm imidazole, and the resulting extract was clarified at 10,000 × g. To encourage complete chromoprotein assembly, the crude soluble extracts were incubated in darkness for 1 h in at least a 10-fold molar excess of BV or protoporphyrin IXa (PPIXa) prior to affinity purification. The holoproteins were then purified via nickel-nitrilotriacetic acid chromatography (Qiagen, Germantown, MD) using 1 m imidazole and 30 mm Tris-HCl (pH 8.0) for elution. The eluate was brought to 0.3 m ammonium sulfate and subjected to phenyl-Sepharose FPLC (GE Healthcare), using 30 mm Tris-HCl (pH 8.0) for elution. Covalent binding of BV to the apoproteins was monitored by zinc-induced fluorescence of the chromoproteins following SDS-PAGE (6Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (245) Google Scholar). Spectrophotometric Analyses—UV-visible absorption spectroscopy was performed with a Lambda 650 spectrophotometer (PerkinElmer Life Sciences). All proteins were diluted with 30 mm Tris-HCl (pH 8.0) so that the Pr absorption maxima at ∼700 nm had an absorbance between 0.25 and 0.6. Spectra of Pr were measured for all proteins following an extended incubation in the darkness. The absorption spectrum of the photoconversion product was obtained immediately following saturating red light (690 nm) irradiation generated using a 10-nm half-bandwidth interference filter (Andover Corp. Salem, NH). Difference spectra were calculated by subtracting the red light-irradiated spectrum from the Pr spectrum. Fluorescence spectra were obtained using a QuantaMaster model C-60/2000 spectrofluorimeter (Photon Technologies International, Birmingham, NJ) with both monochrometers set to a 4-nm band pass. To obtain a more complete excitation spectrum for some of the DrBphP mutants, fluorescence emission was measured at 642 nm in addition to 620 nm. RR spectra were recorded with 1064-nm excitation (Nd-YAG cw laser, line width <1 cm–1) using Digilab Bio-Rad or an RFS 100/S (Bruker Optics, Ettlinger, Germany) Fourier-transform Raman spectrometers (4 cm–1 spectral resolution). The near-infrared excitation line was sufficiently close to the first electronic transition to generate a strong pre-resonance enhancement of the chromophoric vibrational bands, such that Raman bands of the protein matrix remained very weak in the spectra of the parent states (12Kneip C. Hildebrandt P. Schlamann W. Braslavsky S.E. Mark F. Schaffner K. Biochemistry. 1999; 38: 15185-15192Crossref PubMed Scopus (121) Google Scholar, 13von Stetten D. Seibeck S. Michael N. Scheerer P. Mroginski M.A. Murgida D.H. Krauss N. Heyn M.P. Hildebrandt P. Borucki B. Lamparter T. J. Biol. Chem. 2007; 282: 2116-2123Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). PPIXa also did not contribute to the experimental RR data since it does not gain any resonance enhancement with near-infrared excitation. All spectra were measured at –140 °C using a liquid-nitrogen cooled cryostat (Linkam, Waterfield, Surrey, UK). The laser power at the sample was set at ∼700 milliwatts, which did not damage the chromoproteins as checked by comparing the absorption spectra of the samples obtained before and after RR data acquisition. The total accumulation time was less than 2 h for each spectrum. For all RR spectra shown in this work, the background was subtracted manually. Photoconversion intermediates were enriched by irradiating the samples with red light for a few minutes at the specified temperatures. Raw RR spectra measured from these irradiated samples included a substantial contribution from residual Pr, which was removed by subtraction, taking the characteristic RR bands of Pr as a reference. Further RR experimental details have been described previously (12Kneip C. Hildebrandt P. Schlamann W. Braslavsky S.E. Mark F. Schaffner K. Biochemistry. 1999; 38: 15185-15192Crossref PubMed Scopus (121) Google Scholar, 13von Stetten D. Seibeck S. Michael N. Scheerer P. Mroginski M.A. Murgida D.H. Krauss N. Heyn M.P. Hildebrandt P. Borucki B. Lamparter T. J. Biol. Chem. 2007; 282: 2116-2123Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Site-directed modifications of Phys, either randomly or in the context of amino acid sequence alignments, have been widely employed over the past few decades in attempts to identify domains and residues important for Phy assembly, structure, and function (13von Stetten D. Seibeck S. Michael N. Scheerer P. Mroginski M.A. Murgida D.H. Krauss N. Heyn M.P. Hildebrandt P. Borucki B. Lamparter T. J. Biol. Chem. 2007; 282: 2116-2123Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 28Cherry J.R. Hondred D. Walker J.M. Keller J.M. Hershey H.P. Vierstra R.D. Plant Cell. 1993; 5: 565-575PubMed Google Scholar, 29Bhoo S.H. Hirano T. Jeong H.-Y. Lee J.-G. Furuya M. Song P.-S. J. Am. Chem. Soc. 1997; 48: 11717-11718Crossref Scopus (54) Google Scholar, 30Fischer A.J. Lagarias J.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17334-17339Crossref PubMed Scopus (155) Google Scholar, 31Hahn J. Strauss H.M. Landgraf F.T. Gimenez H.F. Lochnit G. Schmieder P. Hughes J. FEBS J. 2006; 273: 1415-1429Crossref PubMed Scopus (68) Google Scholar, 32Stockhaus J. Nagatani A. Halfter U. Kay S. Furuya M. Chua N.H. Genes Dev. 1992; 6: 2364-2372Crossref PubMed Scopus (101) Google Scholar). However, interpreting the direct effects of these changes has been challenging without three-dimensional templates. Here we exploited our high resolution models of D. radiodurans BphP (Protein Data Bank codes 1ZTU, 2O9B, and 2O9C (8Wagner J.R. Zhang J. Brunzelle J.S. Vierstra R.D. Forest K.T. J. Biol. Chem. 2007; 282: 12298-12309Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 11Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (427) Google Scholar)) as guides to directly test the importance of potentially key and often highly conserved amino acids in the CBD with a special focus on those close to the chromophore or central to the knot (Fig. 1, B and C). To minimize secondary effects caused by using truncated chromoproteins, all mutations were introduced into the full-length DrBphP polypeptide with its sequential arrangement of PAS, GAF, PHY, and HK domains. RR analysis revealed that the BV geometry of the unmodified full-length chromoprotein as Pr is nearly identical to that bound to the CBD truncation (supplemental Fig. 1A), 6D. von Stetten, J. R. Wagner, J. Zhang, R. D. Vierstra, and P. H. Hildebrandt, unpublished data. indicating that our CBD models should accurately predict the consequences of the mutations at least for the Pr state. How the mutations might affect Pfr awaits structural resolution of this state. Relative to full-length DrBphP, the CBD chromoprotein becomes trapped in a deprotonated Meta-Rc-like state in red light (supplemental Fig. 1B) (11Wagner J.R. Brunzelle J.S. Forest K.T. Vierstra R.D. Nature. 2005; 438: 325-331Crossref PubMed Scopus (427) Google Scholar), indicating the CBD structure by itself is missing key features for full Pr to Pfr photoconversion. BV assembly was either achieved in vivo following co-expression of the variant BphPs with a HO or in the crude E. coli lysates following the addition of purified BV (6Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (245) Google Scholar). Although these approaches could generate ample quantities of photoactive holoproteins, they precluded quantitative measurement of BV assembly rates. The complete list of variant proteins (38 substitutions/deletions affecting 16 positions), their solubility, ability to assemble with BV, and some of their photochemical and fluorescence characteristics are presented in Table 1.TABLE 1Spectroscopic and photochemical properties of DrBphP variantsConstructionExpression/solubilityBV covalent attachmentPr λmaxRed-irradiated λmaxQ/Soret ratioPhotoconversionPorphyrin fluorescencenmnmWild type+/+Yes7007512.69Pr/PfrNoΔN1-20+/+Yes7017522.34IntermediateNDE25A/E27A+/+Yes6987512.23Pr/PfrNo135A+/+Yes6997512.34IntermediateNDQ36A+/-YesNANANANANAQ36D+/+Yes6987522.47Pr/PfrNDQ36K+/-YesNANANANANAQ36L+/+Yes7017502.62Pr/PfrNDQ36N+/-NANANANANANAP37A+/+Yes7007562.42Pr/PfrNDY176H+/+Yes6967501.28IntermediateNoF203A+/+Yes6967382.13IntermediateNoF203H+/+Yes6957422.16IntermediateNoF203W+/+Yes7017512.37Pr/PfrNoD207A+/+Yes7007511.91IntermediateYesD207E+/+Yes7017512.20IntermediateNoD207H+/+Yes700NA0.83Locked in PrYesD207K+/+Yes700NA1.95Locked in PrYesD207L+/+Yes700NA1.93Locked in PrYesD207N+/+Yes7007511.74IntermediateYesD207Q+/+Yes7017462.02IntermediateYesD207S+/+Yes701NA1.59Locked in PrYesD207T+/+Yes7047511.87IntermediateYesI208A+/+Yes6957512.26Pr/PfrNoP209G+/+Yes6907451.97Pr/PfrNDY216H+/+Yes6977482.42Pr/PfrNoY216W+/+Yes6987510.67IntermediateNDR254A+/+Yes7007501.14Pr/PfrNDR254K+/+Yes7017502.45Pr/PfrNDR254Q+/+NoNANANANANAH260A+/+Yes6987481.16IntermediateNoH260D+/+NoNANANANANAH260K+/+NoNANANANANAH260N+/+Yes6987542.62IntermediateNoH260Q+/+Yes6977482.35Pr/PfrNoH260S+/+Yes6947481.50IntermediateNDY263H+/+Yes6987421.78IntermediateYesH290N+/+Yes6997472.95IntermediateNoH290Q+/+Yes7027462.58IntermediateYes Open table in a new tab Amino" @default.
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• W2069893600 title "Mutational Analysis of Deinococcus radiodurans Bacteriophytochrome Reveals Key Amino Acids Necessary for the Photochromicity and Proton Exchange Cycle of Phytochromes" @default.
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