Human Cryptochrome Light Responsivity Figure 4. Verification of expression of indicated genes by qPCR and consensus promoter elements involved in cry regulation. Solid rectangles represent expression levels in dark, white rectangles expression after two hours of blue light. A: dro3, B: white, C. 7506; D. end. E. Transcription factor binding sequence DNA motifs conserved in cryptochrome regulated promoters of Drosophila as obtained using Drosophila cisTargetX (Aerts et al., 2010), the size of the letter indicates the probability to find the corresponding base at the different positions (adenine (A), cytosine (C), guanine (G) and thymine (T)). doi:10.1371/journal.pone.0031867.g004 length Hscry1 gene was cloned into the pPICZ B Pichia pastoris expression vector without addition of an affinity tag or other heterologous protein sequences. Expression was induced by addition of methanol to yeast cultures (see methods) and verified by Western blot analysis with anti-HsCRY1 antibody to show a single, full length specific protein band (Fig. 5a). Purification was by DEAE column chromatography followed by further fraction- ation over a Resource Q column (see methods). Column fractions containing recombinant HsCRY1 protein at virtual homogeneity were recovered from the purification procedure, as determined by Coomassie staining on SDS polyacrylamide gels (Fig. 5b). Interestingly, upon analysis by UV/Vis spectroscopy, the isolated protein did not show the typical 450 nm peak of oxidized flavin as found in most purified preparations of currently known crypto- chromes, but instead was purified as a red solution whose UV/vis absorption spectrum (Fig. 5c) more closely resembled that of the anionic (‘red’) semiquinone form of flavin [43]. The identity of anionic radical was confirmed by EPR (Fig. 5c inset). In isolated DmCRY and other insect cryptochromes, the anionic species is formed subsequent to illumination and has been correlated with biological activity [9,10], but reverts to the oxidized form upon return to darkness in a matter of minutes. By contrast, the anionic radical is surprisingly stable in Type II HsCRY1, remaining in this oxidation state for weeks (stored at 280uC) and with no obvious spectral change when kept on ice in the dark for several hours. Although certain isolated photolyases can also be purified in the radical form, this is generally as the neutral radical. Since formation of the radical species has been correlated with biological activity [9,12] the apparent stability of this redox form can now provide a possible explanation for the ‘dark active’ role of mammalian type II cryptochromes, which would not need to be reduced by light in order to accumulate the radical form. The isolated purified protein also had a significant peak in the near UV spectral region which could in part be due to flavin (peak at 360 nm) but also may represent some contribution from an antennae (likely folate) cofactor found in most photolyases and some cryptochromes [1,2]. This antenna pigment contributes significant absorption in the near - UV spectral region (peak at 380 nm) in photolyases and its presence in Type II cryptochromes is consistent with reported UV sensitivity of Type II crypto- chromes from butterfly during magnetoreception [44]. To determine the chromophore composition of the protein, the HsCRY1 protein sample was denatured by heating to 95uC and a spectrum taken of the supernatant (Figure S1A). Free flavin appears to have been liberated, showing the typical absorption peaks at 450 nm and at 360 nm. This allowed calculation of the extinction coefficients across the spectral range of the bound anionic semiquinone, based on e = 11.3 mM21 cm21 for liberat- ed, oxidized FAD at 450 nm. The sample in Fig. 5c, therefore, has a radical concentration of ,150 mM (e = 2.66 mM21 cm21 at 478 nm). In addition, the strong peak at around 360 nm had an intensity relative to the 450 nm peak suggesting a possible contribution from 5,10-methenyltetrahydrofolate (MTHF) as well as from FAD (Fig. 5c). This possibility was further explored by fluorescence spectroscopic techniques. As determined by the emission spectrum (excitation at 365 nm), a significant broad peak around 440–460 nm is consistent with the presence of bound folate (Fig. S1B). One of the characteristics of cryptochromes correlated with light sensing is the ability to undergo redox reactions (photoreduction) in response to light. This requires that flavin undergo reduction from a more oxidized form (generally fully oxidized flavin) to a more reduced form (the radical form of flavin is usual for cryptochromes). Such reactions have been detected in vivo; in insect cell cultures expressing high levels of HsCRY1 proteins, photoreduction has been observed by both fluorescence and EPR spectroscopic techniques [9] indicative of oxidized flavin bound to HsCRY1 in vivo. It was therefore to be expected that at least some proportion of the purified protein in vitro should be bound to flavin in the oxidized form, as a condition for possible photoreduction to the (apparently active) radical form. Accord- ingly, to detect the possible presence of oxidized flavin in the sample, the fluorescence excitation spectrum was recorded, monitoring emission at 525 nm (Figure S1C). The resulting spectrum is somewhat broad, which is likely to a result of a combination of factors: the contribution of multiple fluorescing species including that of folate (MTHF); the relatively low extinction coefficient of protein bound flavin in cryptochromes; the relatively low proportion of oxidized flavin bound to HsCRY1. However, there is a clear shoulder at 450 nm, which is consistent with the presence of oxidized FAD in a sample otherwise dominated by the red semiquinone [10,12,13,40]. We next determined whether the purified sample could undergo redox reactions (photoreduction) in response to light. Generally, such reactions are performed in vitro with isolated proteins in the presence of reducing agents such as DTT or 2 -mercaptoethanol [12,13]. An anaerobic sample of HsCRY1 was passed down a 10 DG size-exclusion column (equilibrated with buffer containing 20 mM DTT) to remove any unbound FAD. Spectra where then acquired in the dark and after exposure to 100 mmol m22 s21 blue-light of wavelength 440–460 nm (generated by a cold halogen lamp and a 450 nm bandpass filter). Fig. 5d clearly illustrates that there is a small but significant photoreduction of HsCRY1 under these conditions, with a reduction in absorbance at wavelengths centred around 450 nm (see inset which represents the difference spectrum - dark minus light). The light induced reaction occurred over the course of 85 mins, and presumably represents the reduction of the small amount oxidized flavin bound to the fully reduced form via the anionic semiquinone. The sample was then exposed to air, drawn through a pipette a few times and consequently reoxidized in the dark (within the time taken to aerate the sample and take the spectrum). Interestingly, even at this relatively high degree of illumination, there was almost no reduction in radical flavin, which represents the majority of the bound FAD, to form the fully reduced (FADH-) form typical of photolyases. In this respect, HsCRY1 behaves like plant and Drosophila cryptochromes (which oscillate preferentially between oxidized and radical forms and can not be easily fully reduced in vitro) and unlike photolyases or the photolyase-like Cry-DASH cryptochromes [1]. In summary, HsCRY1 adopts a mixture of flavin redox states upon purification, which contain a large proportion of the anionic radical species that has been correlated with biological activity in PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e31867