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• W2362672802 abstract "Cryptochromes constitute a group of flavin-binding blue light receptors in bacteria, fungi, plants, and insects. Recently, the response of cryptochromes to light was extended to nearly the entire visible spectral region on the basis of the activity of the animal-like cryptochrome aCRY in the green alga Chlamydomonas reinhardtii. This finding was explained by the absorption of red light by the flavin neutral radical as the dark state of the receptor, which then forms the anionic fully reduced state. In this study, time-resolved UV-visible spectroscopy on the full-length aCRY revealed an unusually long-lived tyrosyl radical with a lifetime of 2.6 s, which is present already 1 μs after red light illumination of the flavin radical. Mutational studies disclosed the tyrosine 373 close to the surface to form the long-lived radical and to be essential for photoreduction. This residue is conserved exclusively in the sequences of other putative aCRY proteins distinguishing them from conventional (6–4) photolyases. Size exclusion chromatography showed the full-length aCRY to be a dimer in the dark at 0.5 mm injected concentration with the C-terminal extension as the dimerization site. Upon illumination, partial oligomerization was observed via disulfide bridge formation at cysteine 482 in close proximity to tyrosine 373. The lack of any light response in the C-terminal extension as evidenced by FTIR spectroscopy differentiates aCRY from plant and Drosophila cryptochromes. These findings imply that aCRY might have evolved a different signaling mechanism via a light-triggered redox cascade culminating in photooxidation of a yet unknown substrate or binding partner. Cryptochromes constitute a group of flavin-binding blue light receptors in bacteria, fungi, plants, and insects. Recently, the response of cryptochromes to light was extended to nearly the entire visible spectral region on the basis of the activity of the animal-like cryptochrome aCRY in the green alga Chlamydomonas reinhardtii. This finding was explained by the absorption of red light by the flavin neutral radical as the dark state of the receptor, which then forms the anionic fully reduced state. In this study, time-resolved UV-visible spectroscopy on the full-length aCRY revealed an unusually long-lived tyrosyl radical with a lifetime of 2.6 s, which is present already 1 μs after red light illumination of the flavin radical. Mutational studies disclosed the tyrosine 373 close to the surface to form the long-lived radical and to be essential for photoreduction. This residue is conserved exclusively in the sequences of other putative aCRY proteins distinguishing them from conventional (6–4) photolyases. Size exclusion chromatography showed the full-length aCRY to be a dimer in the dark at 0.5 mm injected concentration with the C-terminal extension as the dimerization site. Upon illumination, partial oligomerization was observed via disulfide bridge formation at cysteine 482 in close proximity to tyrosine 373. The lack of any light response in the C-terminal extension as evidenced by FTIR spectroscopy differentiates aCRY from plant and Drosophila cryptochromes. These findings imply that aCRY might have evolved a different signaling mechanism via a light-triggered redox cascade culminating in photooxidation of a yet unknown substrate or binding partner. Cryptochromes represent a group of diverse sensory photoreceptors present in all kingdoms of life (1.Chaves I. Pokorny R. Byrdin M. Hoang N. Ritz T. Brettel K. Essen L.O. van der Horst G.T. Batschauer A. Ahmad M. The cryptochromes: blue light photoreceptors in plants and animals.Annu. Rev. Plant Biol. 2011; 62: 335-364Crossref PubMed Scopus (585) Google Scholar, 2.Losi A. Gärtner W. The evolution of flavin-binding photoreceptors: an ancient chromophore serving trendy blue-light sensors.Annu. Rev. Plant Biol. 2012; 63: 49-72Crossref PubMed Scopus (144) Google Scholar). Together with the UV-light-dependent DNA repair enzymes, the photolyases (3.Sancar A. Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors.Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1026) Google Scholar), they constitute the cryptochrome/photolyase family. Members of this family share a highly conserved photolyase homology region (PHR) 2The abbreviations used are: PHRphotolyase homology regionFADoxoxidized flavin adenine dinucleotideCCTC-terminal extensionAtCRY1Arabidopsis thaliana cryptochrome 1CPDcyclobutane pyrimidine dimersaCRYanimal-like cryptochromeFADH•FAD neutral radicalFADH−anionic fully reduced state of FADUV-visUV-visibleTrp•tryptophan neutral radicalTyrO•tyrosyl radicalSECsize exclusion chromatographyRNRribonucleotide reductasePSIIphotosystem II. , which comprises ∼500 amino acids and carries a non-covalently bound flavin adenine dinucleotide (FAD) as a chromophore. The C-terminal extension (CCT) present in many cryptochromes and photolyases is strongly variable in amino acid composition and length and has been shown to be crucial for signal transduction in the Arabidopsis cryptochrome AtCRY1 (4.Yang H.Q. Wu Y.J. Tang R.H. Liu D. Liu Y. Cashmore A.R. The C termini of Arabidopsis cryptochromes mediate a constitutive light response.Cell. 2000; 103: 815-827Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). The diverse subfamilies of cryptochromes comprise proteins acting as central blue light sensors in bacteria, fungi, plants, and insects (animal type I CRY) (1.Chaves I. Pokorny R. Byrdin M. Hoang N. Ritz T. Brettel K. Essen L.O. van der Horst G.T. Batschauer A. Ahmad M. The cryptochromes: blue light photoreceptors in plants and animals.Annu. Rev. Plant Biol. 2011; 62: 335-364Crossref PubMed Scopus (585) Google Scholar). Moreover, CRYs are also found as the light-independent, central part of the oscillator of the biological clock in mammals (animal type II CRY) (5.Etchegaray J.P. Lee C. Wade P.A. Reppert S.M. Rhythmic histone acetylation underlies transcription in the mammalian circadian clock.Nature. 2003; 421: 177-182Crossref PubMed Scopus (538) Google Scholar) and as a mediator for light-dependent magnetosensitivity in flies (6.Gegear R.J. Casselman A. Waddell S. Reppert S.M. Cryptochrome mediates light-dependent magnetosensitivity in Drosophila.Nature. 2008; 454: 1014-1018Crossref PubMed Scopus (319) Google Scholar). Opposed to these findings, DASH cryptochromes have been found to repair lesions in single-stranded DNA and double-stranded loop-structured DNA in vitro (7.Pokorny R. Klar T. Hennecke U. Carell T. Batschauer A. Essen L.O. Recognition and repair of UV lesions in loop structures of duplex DNA by DASH-type cryptochrome.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 21023-21027Crossref PubMed Scopus (139) Google Scholar, 8.Selby C.P. Sancar A. A cryptochrome/photolyase class of enzymes with single-stranded DNA-specific photolyase activity.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 17696-17700Crossref PubMed Scopus (246) Google Scholar). Therefore, DASH cryptochromes are more similar in terms of functionality to photolyases than cryptochromes. Among the photolyases, two different types are separated depending on their ability to either repair cyclobutane pyrimidine dimers (CPD) (9.Rupert C.S. Photoreactivation of transforming DNA by an enzyme from bakers' yeast.J. Gen. Physiol. 1960; 43: 573-595Crossref PubMed Scopus (62) Google Scholar) or (6–4) photoproducts (10.Todo T. Takemori H. Ryo H. Ihara M. Matsunaga T. Nikaido O. Sato K. Nomura T. A new photoreactivating enzyme that specifically repairs ultraviolet light-induced (6–4) photoproducts.Nature. 1993; 361: 371-374Crossref PubMed Scopus (259) Google Scholar). Animal type II CRY are closely related to eukaryotic (6–4) photolyases whereas plant cryptochromes are homologous to CPD photolyases. In contrast, prokaryotic (6–4) photolyases (11.Zhang F. Scheerer P. Oberpichler I. Lamparter T. Krauss N. Crystal structure of a prokaryotic (6–4) photolyase with an Fe-S cluster and a 6,7-dimethyl-8-ribityllumazine antenna chromophore.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 7217-7222Crossref PubMed Scopus (76) Google Scholar) and bacterial cryptochromes (12.Geisselbrecht Y. Frühwirth S. Schroeder C. Pierik A.J. Klug G. Essen L.O. CryB from Rhodobacter sphaeroides: a unique class of cryptochromes with new cofactors.EMBO Rep. 2012; 13: 223-229Crossref PubMed Scopus (70) Google Scholar) form a distant subfamily. photolyase homology region oxidized flavin adenine dinucleotide C-terminal extension Arabidopsis thaliana cryptochrome 1 cyclobutane pyrimidine dimers animal-like cryptochrome FAD neutral radical anionic fully reduced state of FAD UV-visible tryptophan neutral radical tyrosyl radical size exclusion chromatography ribonucleotide reductase photosystem II. The paradigm of cryptochromes and other flavoproteins as classical blue light receptors has been challenged recently by the finding that the animal-like cryptochrome (aCRY) from the green alga Chlamydomonas reinhardtii strongly influences gene expression not only in response to blue but also to yellow and red light in vivo (13.Beel B. Prager K. Spexard M. Sasso S. Weiss D. Müller N. Heinnickel M. Dewez D. Ikoma D. Grossman A.R. Kottke T. Mittag M. A flavin binding cryptochrome photoreceptor responds to both blue and red light in Chlamydomonas reinhardtii.Plant Cell. 2012; 24: 2992-3008Crossref PubMed Scopus (120) Google Scholar). These genes code for proteins involved in chlorophyll and carotenoid biosynthesis, light-harvesting complexes, nitrogen metabolism, cell cycle control, and the circadian clock. The extended spectral sensitivity was explained by the presence of the flavin neutral radical (FADH•) in the dark form of the receptor (Fig. 1A) (13.Beel B. Prager K. Spexard M. Sasso S. Weiss D. Müller N. Heinnickel M. Dewez D. Ikoma D. Grossman A.R. Kottke T. Mittag M. A flavin binding cryptochrome photoreceptor responds to both blue and red light in Chlamydomonas reinhardtii.Plant Cell. 2012; 24: 2992-3008Crossref PubMed Scopus (120) Google Scholar) as opposed to the oxidized flavin (FADox) found in plant cryptochromes (14.Banerjee R. Schleicher E. Meier S. Viana R.M. Pokorny R. Ahmad M. Bittl R. Batschauer A. The signaling state of Arabidopsis cryptochrome 2 contains flavin semiquinone.J. Biol. Chem. 2007; 282: 14916-14922Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 15.Bouly J.P. Schleicher E. Dionisio-Sese M. Vandenbussche F. Van Der Straeten D. Bakrim N. Meier S. Batschauer A. Galland P. Bittl R. Ahmad M. Cryptochrome blue light photoreceptors are activated through interconversion of flavin redox states.J. Biol. Chem. 2007; 282: 9383-9391Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). The absorption spectrum of FADH• covers almost the full visible spectrum of light extending up to 680 nm in agreement with the action spectrum of aCRY (13.Beel B. Prager K. Spexard M. Sasso S. Weiss D. Müller N. Heinnickel M. Dewez D. Ikoma D. Grossman A.R. Kottke T. Mittag M. A flavin binding cryptochrome photoreceptor responds to both blue and red light in Chlamydomonas reinhardtii.Plant Cell. 2012; 24: 2992-3008Crossref PubMed Scopus (120) Google Scholar). In vitro, the predominant part of aCRY was found in the oxidized state after purification. Accordingly, formation of FADH• requires a pre-illumination with blue light which was not applied in the in vivo experiments (13.Beel B. Prager K. Spexard M. Sasso S. Weiss D. Müller N. Heinnickel M. Dewez D. Ikoma D. Grossman A.R. Kottke T. Mittag M. A flavin binding cryptochrome photoreceptor responds to both blue and red light in Chlamydomonas reinhardtii.Plant Cell. 2012; 24: 2992-3008Crossref PubMed Scopus (120) Google Scholar). However, FTIR spectroscopy revealed structural changes in the protein moiety only in the transition from FADH• to the anionic fully reduced state (FADH−) of aCRY providing further support for FADH• as the dark state of the chromophore (16.Spexard M. Thöing C. Beel B. Mittag M. Kottke T. Response of the sensory animal-like cryptochrome aCRY to blue and red light as revealed by infrared difference spectroscopy.Biochemistry. 2014; 53: 1041-1050Crossref PubMed Scopus (20) Google Scholar). Strikingly, these changes in turn structures were not detected in the closely related Xenopus laevis (6–4) photolyase (17.Yamada D. Zhang Y. Iwata T. Hitomi K. Getzoff E.D. Kandori H. Fourier-transform infrared study of the photoactivation process of Xenopus (6–4) photolyase.Biochemistry. 2012; 51: 5774-5783Crossref PubMed Scopus (16) Google Scholar). The lifetime of FADH• state in vitro was strongly sensitive to alterations of the pH but not of the oxygen level in contrast to other cryptochromes (18.Berndt A. Kottke T. Breitkreuz H. Dvorsky R. Hennig S. Alexander M. Wolf E. A novel photoreaction mechanism for the circadian blue light photoreceptor Drosophila cryptochrome.J. Biol. Chem. 2007; 282: 13011-13021Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 19.Immeln D. Schlesinger R. Heberle J. Kottke T. Blue light induces radical formation and autophosphorylation in the light-sensitive domain of Chlamydomonas cryptochrome.J. Biol. Chem. 2007; 282: 21720-21728Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 20.Müller P. Ahmad M. Light-activated cryptochrome reacts with molecular oxygen to form a flavin-superoxide radical pair consistent with magnetoreception.J. Biol. Chem. 2011; 286: 21033-21040Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The molecular mechanisms underlying these extraordinary characteristics of aCRY have remained undisclosed up to now because of missing time-resolved information on the early events after red light illumination. Previously, time-resolved UV-visible (UV-vis) spectroscopy on cryptochromes and photolyases has revealed the involvement of transient radical species formed from FAD, tryptophan, and tyrosine as part of the electron transfer cascade (21.Aubert C. Mathis P. Eker A.P. Brettel K. Intraprotein electron transfer between tyrosine and tryptophan in DNA photolyase from Anacystis nidulans.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 5423-5427Crossref PubMed Scopus (141) Google Scholar, 22.Aubert C. Vos M.H. Mathis P. Eker A.P. Brettel K. Intraprotein radical transfer during photoactivation of DNA photolyase.Nature. 2000; 405: 586-590Crossref PubMed Scopus (357) Google Scholar, 23.Giovani B. Byrdin M. Ahmad M. Brettel K. Light-induced electron transfer in a cryptochrome blue-light photoreceptor.Nat. Struct. Biol. 2003; 10: 489-490Crossref PubMed Scopus (240) Google Scholar, 24.Langenbacher T. Immeln D. Dick B. Kottke T. Microsecond light-induced proton transfer to flavin in the blue light sensor plant cryptochrome.J. Am. Chem. Soc. 2009; 131: 14274-14280Crossref PubMed Scopus (83) Google Scholar, 25.Brazard J. Usman A. Lacombat F. Ley C. Martin M.M. Plaza P. Mony L. Heijde M. Zabulon G. Bowler C. Spectro-temporal characterization of the photoactivation mechanism of two new oxidized cryptochrome/photolyase photoreceptors.J. Am. Chem. Soc. 2010; 132: 4935-4945Crossref PubMed Scopus (67) Google Scholar, 26.Müller P. Yamamoto J. Martin R. Iwai S. Brettel K. Discovery and functional analysis of a 4th electron-transferring tryptophan conserved exclusively in animal cryptochromes and (6–4) photolyases.Chem. Commun. 2015; 51: 15502-15505Crossref PubMed Google Scholar, 27.Paulus B. Bajzath C. Melin F. Heidinger L. Kromm V. Herkersdorf C. Benz U. Mann L. Stehle P. Hellwig P. Weber S. Schleicher E. Spectroscopic characterization of radicals and radical pairs in fruit fly cryptochrome - protonated and nonprotonated flavin radical-states.FEBS J. 2015; 282: 3175-3189Crossref PubMed Scopus (30) Google Scholar). Tryptophan neutral radicals (Trp•) exhibit a broad band centered at around 510 nm (28.Solar S. Getoff N. Surdhar P.S. Armstrong D.A. Singh A. Oxidation of tryptophan and N-methylindole by N3., Br2-, and (SCN)2- radicals in light-water and heavy-water solutions - A pulse-radiolysis study.J. Phys. Chem. 1991; 95: 3639-3643Crossref Scopus (149) Google Scholar), whereas tyrosyl radicals (TyrO•) are characterized by two sharp, adjacent bands at 388 and 408 nm (29.Thöing C. Oldemeyer S. Kottke T. Microsecond deprotonation of aspartic acid and response of the α/β subdomain precede C-terminal signaling in the blue light sensor plant cryptochrome.J. Am. Chem. Soc. 2015; 137: 5990-5999Crossref PubMed Scopus (44) Google Scholar, 30.Proshlyakov D.A. UV optical absorption by protein radicals in cytochrome c oxidase.Biochim. Biophys. Acta. 2004; 1655: 282-289Crossref PubMed Scopus (14) Google Scholar). Moreover, the formation of these amino acid radicals in the electron transfer cascade has been disclosed by time-resolved EPR studies (31.Biskup T. Schleicher E. Okafuji A. Link G. Hitomi K. Getzoff E.D. Weber S. Direct observation of a photoinduced radical pair in a cryptochrome blue-light photoreceptor.Angew. Chem. Int. Ed. 2009; 48: 404-407Crossref PubMed Scopus (120) Google Scholar, 32.Weber S. Kay C.W. Mögling H. Möbius K. Hitomi K. Todo T. Photoactivation of the flavin cofactor in Xenopus laevis (6–4) photolyase: observation of a transient tyrosyl radical by time-resolved electron paramagnetic resonance.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1319-1322Crossref PubMed Scopus (76) Google Scholar). However, to date the photoreduction process of the FADH• has not been studied time-resolved in the cryptochrome as well as (6–4) photolyase families. In this study, we focused on resolving the events in aCRY starting from the FADH• state by its selective induction using illumination with red light, because the blue light-induced conversion of FADox in aCRY is not considered to be physiologically relevant (13.Beel B. Prager K. Spexard M. Sasso S. Weiss D. Müller N. Heinnickel M. Dewez D. Ikoma D. Grossman A.R. Kottke T. Mittag M. A flavin binding cryptochrome photoreceptor responds to both blue and red light in Chlamydomonas reinhardtii.Plant Cell. 2012; 24: 2992-3008Crossref PubMed Scopus (120) Google Scholar). To elucidate the red light-induced processes, we employed transient UV-vis spectroscopy, size exclusion chromatography (SEC), and FTIR difference spectroscopy on the full-length aCRY as well as on point mutants Y393A, Y373F, C482A (Fig. 1B), and aCRYΔCCT lacking the 99 residues of the CCT. We identified the contribution from a remarkably long-lived TyrO• formed by Tyr-373 close to the surface (Fig. 1B), which is conserved in aCRY homologues but not in any other subfamily of cryptochromes and photolyases. Furthermore, we investigated the role of the CCT in the light-induced structural response and the oligomerization of aCRY. aCRYΔCCT coding for amino acids 1 to 496 of the aCRY gene, lacking the 99 amino acids of its CCT, was codon-adapted for E. coli (synthesized by Geneart) and cloned into pET28a(+) (Novagen), providing a 6x His-Tag at the C terminus by using the restriction enzyme sites NcoI and HindIII. Mutation Y393A was inserted into the full-length codon-adapted sequence of aCRY (synthesized by Geneart (13.Beel B. Prager K. Spexard M. Sasso S. Weiss D. Müller N. Heinnickel M. Dewez D. Ikoma D. Grossman A.R. Kottke T. Mittag M. A flavin binding cryptochrome photoreceptor responds to both blue and red light in Chlamydomonas reinhardtii.Plant Cell. 2012; 24: 2992-3008Crossref PubMed Scopus (120) Google Scholar)) by the replacement of a 739 bp BstEII and HindIII fragment with a fragment containing the codon-adapted sequence for the substituted amino acid Y393A. Full-length aCRY-Y393A was ligated via NcoI and HindIII restriction sites into vector pET28a(+). The Y373F and C482A mutations were inserted into the full-length aCRY gene in the pET28a(+) vector using phosphorylated back-to-back primers, of which one primer contained the mutation. To amplify the whole plasmid, Phusion DNA polymerase (New England Biolabs) was used in the polymerase chain reaction. The reaction products were ligated afterward. The amino acid exchanges in all resulting plasmids were verified by dideoxy sequencing. Expression and purification of aCRY and its variants were conducted following published procedures (13.Beel B. Prager K. Spexard M. Sasso S. Weiss D. Müller N. Heinnickel M. Dewez D. Ikoma D. Grossman A.R. Kottke T. Mittag M. A flavin binding cryptochrome photoreceptor responds to both blue and red light in Chlamydomonas reinhardtii.Plant Cell. 2012; 24: 2992-3008Crossref PubMed Scopus (120) Google Scholar). Finally, the proteins were obtained in a 50 mm sodium phosphate buffer, pH 7.0, 100 mm NaCl, 20% (v/v) glycerol. The concentration of the sample was adjusted to A447 = 0.3. An HR2000+ spectrometer with DH-2000-BAL light source (Ocean Optics) was used for experiments in the millisecond to second time regime modified with a mesh filter with 35% transmission to avoid sample conversion by the probe light. For illumination, a 451 nm LED (Luxeon Star, Lumileds) with an intensity of 67 milliwatt/cm2 (full width at half maximum (FWHM) of 20 nm) and a 632 nm LED (Luxeon Star, Lumileds) with an intensity of 95 milliwatt/cm2 (FWHM of 15 nm) at the sample were attached to the sample holder perpendicular to the measuring beam. For the generation of FADH•, aCRY was illuminated for 10 s at 451 nm in a 2 × 10 mm fluorescence cuvette (Helma). FADH− was produced by illuminating the sample for 2 s or 10 s at 632 nm. A continuous series of spectra were recorded before, during and after illumination with an integration time of 2 ms and a time resolution of ∼40 ms. Difference spectra were calculated and summarized on a logarithmic time scale to increase the signal-to-noise ratio using MATLAB (The Mathworks). The concentration of the sample was adjusted to A447 = 0.5. The experimental setup for time-resolved UV-vis spectroscopy on slowly recovering systems has been described previously (29.Thöing C. Oldemeyer S. Kottke T. Microsecond deprotonation of aspartic acid and response of the α/β subdomain precede C-terminal signaling in the blue light sensor plant cryptochrome.J. Am. Chem. Soc. 2015; 137: 5990-5999Crossref PubMed Scopus (44) Google Scholar). aCRY was pre-illuminated for 15 s under stirring to generate FADH• using a 455 nm LED (Luxeon Star, Lumileds) with an intensity of 10 milliwatt/cm2 at the sample (FWHM of 20 nm). For excitation of FADH•, a 630 nm pulse with 10 ns duration and 2 mJ/cm2 energy density was generated by a tunable optical parametric oscillator (Opta), which was pumped by the 355-nm third harmonic of a Nd:YAG laser (Quanta-Ray GCR-12, Spectra Physics). Laser pulses with a repetition rate of (1.6 s)−1 were selected by a shutter. Multiple excitations were minimized by rotating a magnetic stirring bar inside the cuvette for 450 ms after each detection. A fast sample exchange within a total volume of 2.5 ml was ensured by a horizontal geometry of the excitation beam. Moreover, contributions from previous excitations were avoided by alternating the recording of reference and signal spectra. All experiments were conducted at 20 °C. Spectra were recorded at 1 μs, 10 μs, 100 μs, and 20 ms after excitation with an integration time of 1 μs, 5 μs, 100 μs, and 100 μs, respectively. Each spectrum was obtained by averaging 10–61 separate experiments, in which each sample was excited 15 times. For the comparison of the wild type and the Y373F mutant of aCRY, ascorbic acid was added as a reducing agent to a final concentration of 3 mm. The samples were pre-illuminated with blue light for 1 s and 45 s for wild type and Y373F mutant, respectively. For excitation, the 532 nm second harmonic of a Nd:YAG laser (Ultra 100, Quantel) was used at a repetition rate of (1.6 s)−1 with a pulse duration of 10 ns and an energy density of 15 mJ/cm2 and 30 mJ/cm2 for wild type and mutant, respectively. The integration time was set to 500 ns for the difference spectra recorded at 500 ns. Each spectrum of aCRY was obtained by averaging 8–20 separate experiments, in which each sample was excited 15 times. The samples were concentrated to an A447 ∼27 by ultrafiltration using Vivaspin 500 filter devices (Sartorius, 50 kDa cutoff). During centrifugation at 15,000 × g, the protein was washed three times with 20 mm sodium phosphate buffer, pH 7.8, 100 mm NaCl, 1% (v/v) glycerol. A 1.8-μl droplet of the sample solution was applied to a BaF2 window (20 mm diameter) and kept at 20 °C and atmospheric pressure for up to 30 s to gently reduce the water content. The samples were sealed with a second BaF2 window. Thus, a well-hydrated film with an absorbance ratio of amide I/water (1650 cm−1) to amide II (1550 cm−1) of 2.3–2.5 was obtained. An appropriate hydration of the sample is essential to ensure that the full extent of changes in secondary structure of the protein is detected. IR experiments were performed on an IFS 66v spectrometer (Bruker) equipped with a photoconductive mercury cadmium telluride (MCT) detector at a spectral resolution of 2 cm−1. The difference spectra were obtained with a long wave pass filter (OCLI) cutting off infrared light above 2256 cm−1. The experiments were performed at 20 °C. The blue light response of aCRY was induced by illumination for 1 s with a 451-nm LED equipped with a diffusion disc and yielding an intensity of 32 milliwatt/cm2 at the sample. Red light illumination was conducted for 10 s with a 632-nm LED with an intensity of 40 milliwatt/cm2 at the sample. To obtain a representative difference spectrum, 512 scans were averaged. SEC was performed using an Äkta purifier (GE Healthcare) with a Superdex200 10/300 GL column (GE Healthcare) at 4 °C. For equilibration and elution, 50 mm phosphate buffer at pH 7.0 and 150 mm NaCl were used. Aliquots of 100 μl with a protein concentration of 0.5 mm were centrifuged at 21,400 × g for 10 min at 4 °C. For the investigation of aCRY carrying FADox and FADH•, loading of the samples and the SEC were performed in the dark. FADH• was obtained by illuminating the sample for 10 s with two 451 nm LEDs (Luxeon Star, Lumileds) with an intensity of 67 milliwatt/cm2 each at the sample. The elution profiles were recorded at 447 and 630 nm to ensure that only protein with the flavin bound contributes. For the generation of FADH−, samples were illuminated for 45 s with the two 451 nm LEDs followed by 30 s in darkness and illumination for 20 s with a 451 nm LED and a 632 nm LED with an intensity of 64 milliwatt/cm2. Alternatively, the latter two LEDs were used to illuminate the sample for 45 s. This variation in illumination did not have any detectable effect on the elution profiles of the proteins recorded at 447 and 370 nm. Standard marker proteins (GE Healthcare) were used to determine the apparent molecular mass of the sample by calibration. Representative traces are shown from the experiments, which were repeated at least three times for each of 2–3 independent preparations. Phylogenetic analyses were conducted using the MEGA software package version 6 (33.Tamura K. Dudley J. Nei M. Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0.Mol. Biol. Evol. 2007; 24: 1596-1599Crossref PubMed Scopus (25548) Google Scholar). The protein sequences used for the alignment were selected according to the results of the NCBI BLASTP 2.3.0 (34.Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59759) Google Scholar) using the sequences with the highest identity to aCRY. Additionally, sequences of closely related and well characterized cryptochromes and photolyases were included (supplemental Table S1). The protein sequences were aligned with the ClustalW algorithm. The UV-vis spectrum of aCRY recorded after purification revealed FADox to be present in aCRY in vitro (Fig. 1C). Blue light illumination then generated FADH• with characteristic bands at 585 and 633 nm. We noticed an additional small band at 416 nm with a shoulder at 410 nm in the absorption spectrum after illumination, which was only present for a few seconds (Fig. 1C). Such contribution is indicative for transient accumulation of an amino acid radical, but usually detected on a much shorter time scale. Therefore, this finding was scrutinized in a time-resolved manner in the context of the physiologically more relevant reduction of FADH• to FADH−. Time-resolved UV-vis experiments have revealed the involvement of tryptophan and tyrosyl radicals in the photoreaction of cryptochromes in vitro starting from FADox. Here, flash photolysis was applied to detect possible amino acid radicals involved in the red light-induced photoreduction of FADH• in aCRY. Previous steady-state experiments have shown only the formation of FADH− (16.Spexard M. Thöing C. Beel B. Mittag M. Kottke T. Response of the sensory animal-like cryptochrome aCRY to blue and red light as revealed by infrared difference spectroscopy.Biochemistry. 2014; 53: 1041-1050Crossref PubMed Scopus (20) Google Scholar). Samples containing FADox were pre-illuminated with blue light for 15 s to generate a sufficient amount of FADH•, which was stabilized by adjusting the pH to 7.0 (16.Spexard M. Thöing C. Beel B. Mittag M. Kottke T. Response of the sensory animal-like cryptochrome aCRY to blue and red light as revealed by infrared difference spectroscopy.Biochemistry. 2014; 53: 1041-1050Crossref PubMed Scopus (20) Google Scholar). To selectively convert FADH• to FADH−, nanosecond laser pulses at 630 nm were used. Time-resolved UV-vis difference spectra were recorded at time points from 1 μs to 2 ms (Fig. 2A). All difference spectra show the characteristic negative band pattern of the bleaching of FADH• with two broad maxima at 590 nm and 630 nm. Additionally, a small positive contribution from the formation of FADH− is" @default.
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• W2362672802 date "2016-07-01" @default.
• W2362672802 modified "2023-10-14" @default.
• W2362672802 title "Essential Role of an Unusually Long-lived Tyrosyl Radical in the Response to Red Light of the Animal-like Cryptochrome aCRY" @default.
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