harvested after 48 hours by centrifugation, resuspended in 50 mM potassium phosphate pH 7.5, 20% glycerol, 0.05% Triton X-100, 40 mM FAD, 1 mM PMSF, and lysed by passing twice through a cell disruptor (TS-series, 1.1 kW, Constant Systems Ltd) at 15,000 psi. The supernatant was clarified by centrifugation and loaded on a DEAE-sepharose column equilibrated with Buffer A: 50 mM potassium phosphate pH 7.5, 10% glycerol and 0.05% Triton X- 100. The column was eluted with a linear gradient of Buffer A containing KCl from 0 to 300 mM. Fractions containing Hscry1 where pooled, dialysed against Buffer A overnight, and loaded onto a 6 ml Resource-Q column. This column was eluted against a linear gradient of Buffer A containing 0 to 500 mM KCl, and the fractions of purified HsCRY1 (SDS PAGE, Figure 1b) were dialysed once more against buffer A to remove the salt. The sample was concentrated by ultrafiltration through a 30,000 MWCO PES membrane. EPR Analysis Solutions of HsCRY1 (250 ml, ,290 mM) in 4 mm o.d. suprasil quartz tubes (Wilmad, USA) were frozen to 77 K in liquid nitrogen. The tubes were then transferred to a Bruker E500/E580 X-band EPR spectrometer equipped with a Super High Q (SHQ) resonator wherein sample temperature was maintained at 80 K using an Oxford Instruments ESR900 cryostat coupled to an ITC503 Intelligent Temperature Controller from the same manufacturer. Accurate g-values were determined using a Bruker NMR Gaussmeter. Spectra were obtained in continuous wave mode with a microwave power of 10 mW, a field modulation frequency of 100 kHz and a modulation amplitude of 1 G. References 1. Chaves I, Pokorny S, Byrdin M, Hoang N, Ritz T, et al. (2011) The cryptochromes: blue light photoreceptors in plants and animals. Ann Review Plant Biology 62: 335–64. 2. Sancar A (2004) Photolyase and cryptochrome blue-light photoreceptors. Adv Protein Chem 69: 73–100. 3. Lin C, Todo T (2005) The cryptochromes. Genome Biol 6: 220. 4. Tamanini F, Chaves I, Bajek MI, van der Horst GT (2007) Structure function analysis of mammalian cryptochromes. Cold Spring Harb Symp Quant Biol 72: 133–9. 5. Busza A, Emery-Le M, Rosbash M, Emery P (2004) Roles of the two Drosophilla cryptochrome structural domains in circadian photoreception. Science 304: 1503–6. 6. Chaves I, Yagita K, Barnhoorn S, Okamura H, van der Horst GT, et al. (2006) Functional evolution of the photolyase/cryptochrome protein family: impor- tance of the C terminus of mammalian CRY1 for circadian core oscillator performance. Mol Cell Biol 26: 1743–53. 7. Liu B, Liu H, Zhong D, Lin C (2010) Searching for a photocycle of the cryptochrome photoreceptors. Current Opin Plant Biology 13: 578–586. 8. Van Vickle-Chavez SJ, Van Gelder RN (2007) Action spectrum of Drosophila cryptochrome. J Biol Chem 282: 10561–10566. 9. Hoang N, Schleicher E, Kacprzak S, Bouly J-P, Picot M, et al. (2008) b Human and Drosophila cryptochromes are light activated by flavin photoreduction in living cells. PLoS Biology 6: e160. 10. Berndt A, Kottke T, Breitkreuz H, Dvorsky R, Hennig S, et al. (2007) A novel photoreaction mechanism for the circadian blue light photoreceptor Drosophila cryptochrome. J Biol Chem 282: 13011–13021. 11. Song SH, Ozturk N, Denaro TR, Arat NO, Kao YT, et al. (2007) Formation and function of flavin anion radical in cryptochrome 1 blue-light photoreceptor of monarch butterfly. J Biol Chem 282: 17608–17612. 12. Bouly JP, Schleicher E, Dionisio-Sese M, Vandenbussche F, Van Der Straeten D, et al. (2007) Cryptochrome blue light photoreceptors are activated through interconversion of flavin redox states. J Biol Chem 282: 9383–9391. 13. Banerjee R, Schleicher E, Meier S, Viana RM, Pokorny R, et al. (2007) The signaling state of Arabidopsis cryptochrome 2 contains flavin semiquinone. J Biol Chem 282: 14916–14922. 14. Krishnan B, Levine JD, Lynch MK, Dowse HB, Funes P, et al. (2001) A new role for cryptochrome in a Drosophila circadian oscillator. Nature 411: 313–7. 15. Collins B, Mazzoni EO, Stanewsky R, Blau J (2006) Drosophila cryptochrome is a circadian transcriptional repressor. Curr Biol 16: 441–9. 16. Ivanchenko M, Stanewsky R, Giebultowicz JM (2001) Circadian photoreception in Drosophila: functions of cryptochrome in peripheral and central clocks. J Biol Rhythms 16: 205–15. Human Cryptochrome Light Responsivity Supporting Information Figure S1 A - The UV-vis absorbance spectrum of purified HsCRY1 is shown in the solid line. The dashed line, which shows the absorbance spectrum of supernatant after thermally denatur- ing HsCRY1 by heating to 95uC for 10 mins and centrifugation, was used to calculate the extinction coefficient. B - Fluorescence emission spectrum of purified HsCRY1 upon excitation at 365 nm. The broad peak between 440–460 nm is consistent with the presence of folate cofactor. C - Fluorescence excitation spectra of HsCRY1 monitored at an emission wavelength of 525 nm. The shoulder at 450 nm is consistent with the presence of a small quantity of oxidized flavin. (TIFF) Acknowledgments Thanks to Dr. Stephen E. J. Rigby for help with the EPR spectroscopy. Nigel Scrutton is a BBSRC Professorial Research Fellow and a Royal Society Wolfson Merit Award holder. Alex Jones is a Colt Foundation Postdoctoral Research Fellow. Many thanks to B. Sotta, S. Paumard, V. Marre´chal, Kim, T. Foulon and E. Miginiac for valuable assistance and advice. Thanks also to Jeffrey Steward and Tara C. Ambrosine for help with experiments. Author Contributions Conceived and designed the experiments: JV ARJ AD MS NH DR ST DJH MP TY GS JYC AK FR NS MA. Performed the experiments: JV ARJ AD MS NH DR ST DJH MP TY GS JYC AK MA. Analyzed the data: JV ARJ AD MS NH DR ST DJH MP TY GS JYC AK FR NS MA CH. Contributed reagents/materials/analysis tools: GS JYC. Wrote the paper: JV ARJ AD GS JYC FR NS MA CH. 17. van der Horst GT, Muijtjens M, Kobayashi K, Takano R, Kanno S, et al. (1999) Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature 398: 627–30. 18. Okamura H, Miyake S, Sumi Y, Yamaguchi S, Yasui A, et al. (1999) Photic induction of mPer1 and mPer2 in Cry-deficient mice lacking a biological clock. Science 286: 2531–4. 19. Vitaterna MH, Selby CP, Todo T, Niwa H, Thompson C, et al. (1999) Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2. Proc Nat Acad Sci USA 96: 12114–9. 20. Kume K, Zylka MJ, Sriram S, Shearman LP, Weaver DR, et al. (1999) mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98: 193–205. 21. Griffin EA, Jr., Staknis D, Weitz CJ (1999) Light-independent role of CRY1 and CRY2 in the mammalian circadian clock. Science 286: 768–771. 22. Yagita K, Tamanini F, van Der Horst GT, Okamura H (2001) Molecular mechanisms of the biological clock in cultured fibroblasts. Science 292: 278–81. 23. Tamanini F (2007) Manipulation of mammalian cell lines for circadian studies. Methods Mol Biol 362: 443–53. 24. Zhu H, Yuan Q, Briscoe AD, Froy O, Casselman A, et al. (2005) The two CRYs of the butterfly. Curr Biol 6: R953–4. 25. Gegear RJ, Foley LE, Casselman A, Reppert SM (2010) Animal cryptochromes mediate magnetoreception by an unconventional photochemical mechanism. Nature 463: 804–7. 26. Foley LE, Gegear RJ, Reppert SJ (2011) Human cryptochrome exhibits light- dependent magnetosensitivity. Nature Communications 2: 356. | DOI: 10.1038. 27. Rubin GM, Hazelrigg T, Karess RE, Laski FA, Laverty T, et al. (1985) Germ line specificity of P-element transposition and some novel patterns of expression of transduced copies of the white gene. Cold Spring Harb Symp Quant Biol 50: 329–35. 28. Stanewsky R, Kaneko M, Emery P, Beretta B, Wager-Smith K, et al. (1998) The cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila. Cell 95: 681–92. 29. Kaneko M (1998) Neural substrates of Drosophila rhythms revealed by mutants and molecular manipulations. Curr Opin Neurobiol 8: 652–658. 30. Yu X, Shalitin D, Liu X, Maymon M, Klejnot J, et al. (2007) C. Derepression of the NC80 motif is critical for the photoactivation of Arabidopsis CRY2. Proc Natl Acad Sci USA 104: 7289–94. 31. Emery P, Stanewsky R, Hall JC, Rosbash M (2000) A unique circadian-rhythm photoreceptor. Nature 404: 456–7. 32. Dolezelova E, Dolezel D, Hall JC (2007) Rhythm defects caused by newly engineered mutations in Drosophila’s cryptochrome gene. Genetics 177: 329–45. PLoS ONE | www.plosone.org 13 March 2012 | Volume 7 | Issue 3 | e31867