FEBS Letters
Volume 583, Issue 9 , Pages 1427-1433, 6 May 2009

Conformational change induced by ATP binding correlates with enhanced biological function of Arabidopsis cryptochrome

Edited by Richard Cogdell

  • Sarah Burney

      Affiliations

    • Université Paris 6, CNRS – UMR 7180, PCMP, F-75005 Paris, France
    • ,
  • Nathalie Hoang

      Affiliations

    • Université Paris 6, CNRS – UMR 7180, PCMP, F-75005 Paris, France
    • ,
  • Michael Caruso

      Affiliations

    • Penn State University, Media, PA, USA
    • ,
  • Elizabeth A. Dudkin

      Affiliations

    • Penn State University, Media, PA, USA
    • ,
  • Margaret Ahmad

      Affiliations

    • Université Paris 6, CNRS – UMR 7180, PCMP, F-75005 Paris, France
    • Penn State University, Media, PA, USA
    • ,
  • Jean-Pierre Bouly

      Affiliations

    • Université Paris 6, CNRS – UMR 7180, PCMP, F-75005 Paris, France
    • Corresponding Author InformationCorresponding author. Address: Université Paris 6, CNRS – UR5, PCMP, Casier 156, 4 Place Jussieu, 75005 Paris, France. Fax: + 33 144272916.

Received 27 January 2009; received in revised form 17 March 2009; accepted 18 March 2009. published online 25 March 2009.

Article Outline

Abstract 

Cryptochromes are widely distributed blue light photoreceptors involved in numerous signaling functions in plants and animals. Both plant and animal-type cryptochromes are found to bind ATP and display intrinsic autokinase activity; however the functional significance of this activity remains a matter of speculation. Here we show in purified preparations of Arabidopsis cry1 that ATP binding induces conformational change independently of light and increases the amount and stability of light-induced flavin radical formation. Nucleotide binding may thereby provide a mechanism whereby light responsivity in organisms can be regulated through modulation of cryptochrome photoreceptor conformation.

Keywords: Cryptochrome, Photolyase, Blue light, Photoreceptor, ATP, Arabidopsis thaliana

 

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1. Introduction 

Cryptochrome blue light photoreceptors are found throughout the biological kingdom including in plants, animals and humans. Cryptochromes show significant structural similarity to photolyases, a class of light-driven DNA repair enzyme that repairs Cyclobutane Pyrimidine Dimer (CPD) and 6-4 type lesions in UV-damaged DNA. Unlike photolyases, most cryptochromes do not efficiently repair DNA and have instead evolved novel roles in signaling, including blue light dependent developmental processes in plants and the entrainment of the circadian clock in animals [1], [2]. Interestingly, cryptochromes apparently arose multiple times during the course of evolution from different photolyase ancestors. For instance plant cryptochromes as defined by the Arabidopsis cry1 and cry2 gene family are more similar to microbial type I CPD photolyases whereas animal-type cryptochromes as defined by mammalian and insect cryptochromes are more similar to the eukaryotic 6-4 type photolyases [3]. Despite their differing evolutionary origins, there is recently considerable evidence that plant and animal-type cryptochromes are activated by a mechanism involving electron transfer and flavin radical formation [4], [5], [6], [7], [8], [9], [10]. This mechanism is distinct from that occurring in photolyases, where flavin is in the fully reduced form for the enzyme to be catalytically active in vivo.

An additional difference in the properties of cryptochromes as compared to photolyases is an ATP binding and autokinase activity present in purified preparations of plant cryptochromes from Arabidopsis [11], [12], [13] and Chlamydomonas [14], as well as human Hscry1 [11], [13] and Hscry2 [13]. The ATP binding site has been located within the cavity proximal to flavin in a region that is homologous to the DNA binding pocket in photolyase [15]. It has been shown for Chlamydomonas cryptochrome that ATP binding favors radical accumulation by increasing the lifetime of this redox form during dark reoxidation. These changes are in agreement with a possible functional role in vivo, as the signaling state of plant cryptochromes has been correlated with radical accumulation [6], [7], [14]. In the present work, we extend these observations to Arabidopsis cry1 and show that ATP binding favors both the rate and extent of radical accumulation and its stability upon dark reversion in the presence of light. We furthermore provide evidence of a conformational change that occurs independently of light upon ATP binding of Arabidopsis Atcry1. Taken together, these results suggest nucleotide binding may be a novel form of regulation of cryptochrome activity both together with/or independently of light.

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2. Materials and methods 

2.1. Purification of the recombinant proteins 

To purify cryptochromes, Sf21 cells (1×106cells/ml) were inoculated with the appropriate baculovirus and incubated at 27°C. After 48h, the cells are spin down at 1500rpm and lysed in a lysis buffer (50mM Tris–HCl at pH 7.5, 1% Triton X-100 for 60min on ice. The cell lysate is spin down at 15000rpm and the supernatant is incubated with NTA resin (Qiagen) equilibrated with lysis buffer at 4°C for 1h. The resin is wash 1 time with wash buffer A (50mM Tris–HCl at pH 7.5, 150mM NaCl, and 10% glycerol), 1 time with wash buffer B (50mM Tris–HCl at pH 7.5, 300mM NaCl, at pH 8.0, and 10% glycerol) and 1 time with wash buffer C (50mM Tris–HCl at pH 7.5, 300mM NaCl, at pH 8.0; 5mM imidazole and 10% glycerol). The cryptochromes are eluted with elution buffer (10mM Tris–HCl at pH 7.4, 150mM NaCl, and 250mM imidazole). Salt and imidazole are remove through overnight dialysis against dialysis buffer (50mM Tris–HCl at pH 7.5, 100mM NaCl, and 20% glycerol). The purity of the proteins is checked by SDS–PAGE and Coomassie blue staining and has been estimated between 90% and 98% of homogeneity. The flavin content is estimated by absorption spectroscopy using the FAD molar extinction coefficient at 450nm . Absorption spectra were recorded using a cary 300 scan (Varian) spectrophotometer.

2.2. Photoreduction and oxidation 

Cryptochromes (wild type, W400F and W324F [5]) in dialysis buffer are irradiated with 450nm light at 50 or 100μmolm−2s−1. Photoreduction, oxidation and spectrum have performed at 12°C. Absorption spectra were recorded using a cary 300 scan (Varian) spectrophotometer. Some photoreduction experiments were carried out with 10mM β-mercaptoethanol and under aerobic or anaerobic conditions.

2.3. Fluorescence experiments 

Cryptochromes (wild type, W400F, and W324F) in dialysis buffer are incubated 10min at 4°C over a range of different ATP concentration in the dark. Fluorescence emission and excitation was monitored in a Varian cary eclipse fluorescence spectrophotometer over a range of excitation wavelengths or emission wavelength as indicated.

2.4. Trypsin experiments 

For flavin release, 100μM of full-length Atcry1 were digested in 50mM Tris–HCl, pH 7.5 and 0.125ng/μl of trypsin at 30°C in the dark directly in the spectrofluorometer with or without 1mM ATP. The emission of the flavin (excitation at 445mm and emission at 525nm) has been continuously recorded. For trypsin digestion kinetics equal quantities of full-length Atcry1 (20μM) were digested in 25mM NH4HCO3 and 0.125ng/μl of trypsin at 25°C in the dark. Samples with 1mM of ATP have been preincubated at 4°C with ATP during 15min. The reactions were stopped at 15, 30, 60 and 120min with 5X SDS buffer, resolved by 12.5% SDS–PAGE and visualized by Coomassie staining.

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3. Results 

3.1. Addition of ATP increases levels of FADH° product formation subsequent to Atcry1 photoreduction 

We investigated the effect of addition of ATP on Atcry1 blue light activation. We found that addition of ATP in the presence of MgCl2, which are the conditions determined to stimulate autophosphorylation activity [11], [12], [13], [14], resulted in significantly increased levels of photoreduced FADH° as compared to Atcry1 in the absence of ATP (Fig. 1A–C). Saturation was reached after about 6min illumination under all conditions. Light-induced FADH° accumulation was similarly enhanced by added ATP in the absence of MgCl2 or in the presence of the non-hydrolysable AMP-PnP analog (Fig. 1D and E). Since neither of these conditions promotes autophosphorylation, these results shows that ATP has an effect on the photoreduction of cryptochrome without any requirement for phosphorylation, and that this effect is essentially due to the binding of ATP on cryptochrome. Interestingly, no effect of AMP has been detected on the photoreduction reaction (Fig. 1F). In order to determine if AMP does not bind to cryptochrome or if the binding of AMP does not lead to the increased of photoreduction, we have done competition experiments between ATP and AMP. These experiments show that AMP does not compete with ATP for enhancement of FADH° product formation (Fig. 1F) due to a much lower or absent AMP binding affinity for cryptochrome.

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  • Fig. 1. 

    Kinetics of difference spectra of cryptochrome 1 (10μM) photoreduced by exposure at 100μmolm−2s−1 of 450nm light during 1, 2, 4 and 6min and under aerobic conditions without reducing agent. (A) Cryptochrome 1 of Arabidopsis thaliana, (B) cryptochrome 1 of A. thaliana and 1mM MgCl2, (C) cryptochrome 1 of A. thaliana and 0.2mM ATP and 1mM MgCl2, (D) cryptochrome 1 of A. thaliana and 0.2mM ATP, (E) cryptochrome 1 of A. thaliana and 0.2mM AMP-PnP, (F) difference spectra of cryptochrome 1 photoreduced after 6min of light without nucleotide, with AMP (1mM), with ATP (1mM), with AMP (1mM) and ATP (1mM), with AMP (10mM) and ATP (1mM).

In both photolyases and cryptochromes, a triad of tryptophan residues mediating intraprotein electron transfer from the protein surface to the excited state flavin has been shown to be the mechanism for photoreduction. Mutations of W324 (surface-exposed) and W400 (proximal to the flavin) of Arabidopsis cryptochrome to the redox inactive phenylalanine (F) residue results in a significant decrease in in vitro photoreduction of the protein and reduced biological activity in transgenic plants [5]. We have tested the effects of ATP on in vitro photoreduction in these two mutant proteins to determine whether the effect of ATP on FADH° accumulation requires a functional electron transfer pathway. Fig. 2 shows that both mutations W324F and W400F blocked photoreduction of FADox to FADH°. In the presence of 1mM ATP, no difference has been observed in the W324F mutant and only a small increase in photoreduction is observed in the mutant W400F. These results suggest that the increased photoreduction of cryptochrome observed in the presence of ATP is still dependant on the tryptophan triad pathway for electron transfer and not some novel “ATP dependant electron transfer pathway”.

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  • Fig. 2. 

    Photoreduction of Atcry1 (10μM), W400F (10μM), and W324F (10μM) by exposure during 30s at 100μmolm−2s−1 of 450nm light under aerobic condition in the presence or absence of ATP.

3.2. ATP binding significantly slows the rate of Atcry1 reoxidation in the dark 

The steady state level of FADH° achieved in the presence of ATP can be due both to a higher efficiency of photoreduction or an increased stability of the radical. Therefore, we have tested the rate of dark reoxidation of Atcry1 in the presence or absence of ATP. The samples were illuminated with blue light for 30s under aerobic conditions. After 30s of illumination, the presence of the fully reduced form is undetectable and only a mix of FAD ox and FADH° is observed (Fig. 3). The sample was then placed in darkness and recovery of the oxidized state was measured in the presence of oxygen. Addition of ATP to the sample strongly reduced the rate of reoxidation of Atcry1 as complete reoxidation of flavin was reached after 5min in the absence of ATP and more than 70min in the presence of ATP (Fig. 3). As both oxidation reactions followed a mono exponential shape, we calculated a time constant of 2.6min for reoxidation without ATP and 15.3min for samples in the presence of ATP.

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  • Fig. 3. 

    Reoxidation kinetics of photoreduced Atcry1. Atcry1 (10μM), were reduced with 100μmolm−2s−1 of 450nm light during 1min and kept in the dark under aerobic conditions with 10mM of β-mercaptoethanol with 1mM of AMP-PnP (diamond) or without (triangle). O.D. at 450nm to follow the fully oxidized form are plotted in the graph. The difference spectrum observed are indicated in the two insets.

These results suggest that ATP binding significantly slows the rate of reoxidation and in this way stabilizes the levels of FADH°, providing an explanation for the considerably enhanced FADH° accumulation in response to light. However, it cannot be ruled out from these data that ATP binding also enhances the forward rate of photoreduction. The relative amount of FADH° product formed after even 1min illumination in the presence of ATP is close to 10-fold higher than that of samples in the absence of ATP (Fig. 1). This level of increase cannot be fully explained by the differential in reoxidation rate on a 1min time scale.

3.3. ATP binding significantly reduces trp fluorescence quenching indicative of conformational change in Atcry1 

Fluorescence of tryptophan in proteins is dependant on their environment. Consequently the variation in the tryptophan fluorescence of a protein provides information about changes in conformation. The emission spectra for cryptochrome fluorescence were recorded at an excitation wavelength of 285nm to follow tryptophan fluorescence and at 445nm to follow flavin fluorescence, respectively. Spectra were compared in samples with and without the addition of 1mM ATP without illumination or photoreduction. As shown in Fig. 4, ATP binding resulted in a decreased fluorescence of both the tryptophan and the flavin without affecting the position of the emission peaks, thereby suggesting that no denaturation occurs. The decrement in the fluorescence intensity observed in the presence of ATP was from in a saturated fashion with increasing ATP concentration up to 1mM for tryptophan and for flavin emission. The ATP concentration corresponding to 50% quenching (Kd) was 200±18μM, which is higher but not dissimilar to the Kd values from 4 to 75μM observed previously for cryptochromes [11], [13], [14]. The effect of ATP binding on the photoreduction-defective mutant proteins W324F and W400F was also tested. On the assumption that these W substitution mutations might by themselves affect tryptophan emission characteristics, we only tested for changes in the fluorescence of FAD. The wavelength giving the emission maxima with excitation at 445nm of W324F and W400F were the same as that of the wild type suggesting that these mutants are not altered in conformations as compared to wild type. In the presence of ATP there was a decrease in fluorescence of both mutant consisting of a for W400F and of 17.3±4% for W324F. These data strongly suggest structural changes in Atcry1 upon ATP binding. Furthermore, these apparent structural changes are triggered directly by ATP binding to the photoreceptor. They do not require light or any light dependent activation reaction such as electron transfer and subsequent flavin photoreduction. However, it is also possible that the conformational change induced by ATP binding may improve electron transfer efficiency from trp to FAD induced by light.

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  • Fig. 4. 

    (A) Fluorescence of Atcry1 (10μM), in the presence of different ATP concentration excitation at 285nm, emission at 370nm have been used to follow the tryptophan fluorescence. Excitation at 445nm, emission at 525nm have been used to follow the FAD fluorescence. Emission are recorded after 10min of incubation with ATP. (B) Excitation or emission spectra of Atcry1 (A), W400F (B) and W350F (C) with or without ATP. Emission 18 spectra have been recorded with light excitation at 445nm. Excitation spectra have been recorded with an emission at 525nm.

3.4. ATP binding renders Atcry1 more resistant to denaturation by guanidine consistent with conformational change 

In order to extend the observations derived from fluorescence changes in the protein, an independent method of testing for conformational change was applied. Guanidine chloride is a commonly used denaturant that can unfold native proteins, which can be rendered more or less resistant to unfolding depending on the conformation that they adopt [16]. We have probed possible ATP-induced conformational changes in Atcry1 that may alter the resistance of the protein to this denaturant. Unfolding of Atcry1 was followed spectroscopically using intrinsic FAD fluorescence as a marker. FAD is bound in a hydrophobic pocket in the interior of the protein and fluorescence is considerably quenched in the folded protein [4], [15]. Upon unfolding of the protein, flavin is released into solution resulting in a significant increase of the FAD fluorescence (Fig. 4A). Increases in FAD fluorescence were monitored at concentrations of GnCl ranging from 0 to 4M and unfolding monitored using increased flavin fluorescence. Unfolding accordingly occurred maximally between 1 and 2M concentrations of GnCl. In a parallel experiment, an identical sample with the addition of 1mM ATP was treated in a similar manner with increasing concentrations of GnCl. In this case however, unfolding occurs consistently at higher GnCl concentrations over the entire time course of the unfolding reaction. This result showed that ATP binding induced increased stabilization against the denaturing effect of guanidine chloride on Atcry1 and thereby provides support for the likelihood of conformational changes altering protein stability. These effects occur in the dark, without the requirement for irradiation of flavin photoreduction to become manifest, and are thereby consistent with the effects on W fluorescence quenching described above (Fig. 5).

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  • Fig. 5. 

    Unfolding of Atcry1 in the presence of GndCl. Atcry1 (10μM) has been incubated with increasing concentration of GdnCl. Flavine fluorescence (excitation 445nm–emission 525nm) was recorded until the steady state was reached.

3.5. ATP binding alters the sensitivity of Atcry1 to proteolysis by trypsin 

Differential proteolysis is a widely used and accepted method to probe conformational change in proteins based on altered accessibility of proteolytic cleavage sites [17], [18], [19]. We accordingly probed the proteolytic sensitivity of Atcry1 to trypsin by two independent methods. In the first approach, slow proteolysis of Atcry1 by trypsin was performed over time and the samples assayed for increased flavin fluorescence as an indication of denaturation and flavin release. In this experiment, trypsin was added to identical samples either in the presence or absence of 1mM ATP. Limiting concentrations of trypsin were added and the samples placed in the fluorometer at intervals. Emission spectra were obtained at 525nm subsequent to excitation at 445nm, which excites oxidized flavin. A kinetic plot of flavin release (Fig. 6A) shows that the ATP-bound cryptochrome form exhibited a higher resistance to release the flavin into the solution than did the control without ATP. After 4h the in the absence of ATP and in the presence of ATP. These data indicate that the ATP binding renders the cryptochrome more resistant to proteolysis and is thereby consistent with an induced conformational change.

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  • Fig. 6. 

    (A) Comparative analysis of trypic digest of Atcry1 with or without ATP. Kinetic of release of free flavin. Emission spectra at 525nm have been recorded with light excitation at 445nm. (B) Kinetics of limited proteolysis assays. The reactions were stopped at 15, 30, 60, and 120min and Atcry1 species were separated using 12% SDS–PAGE and then coomassie blue stained.

In a second approach, the pattern of proteolysis by trypsin was measured under the same conditions by partial proteolysis followed by SDS–PAGE resolution of digested proteins. Interestingly, cryptochrome 1 bound to ATP was cleaved into fragments with different apparent sizes on coomassie stained SDS–PAGE than were unbound samples (Fig. 6B). In the absence of ATP, peptides of a 10 and 12kDa (band a and b) and a faster hydrolysis of a 30kDa peptide (band 1) are visible whereas the 15 and 30kDa peptides are more stable in the presence of ATP (band 1 and 2). Moreover, a peptide of 23kDa (band x) shows the same kinetic in presence or absence of ATP. These results show that ATP binding modifies not only the kinetics but also the tryptic pattern of Atcry1 and, taken together with the experimental results on trp fluorescence quenching and differential protein denaturation by GnCl, provide powerful evidence for induced conformational change.

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4. Discussion 

In this study, we have demonstrated that binding of ATP to Arabidopsis cryptochrome increases the rate and the extent of photoreduction in response to blue light irradiation. In this respect our results are in good agreement with those reported previously for Chlamydomonas cryptochrome, including the fact that subsequent autokinase activity is not required in this activation [14]. These effects of ATP binding on Atcry1 photoreduction correlate very well with a functional role in mediating biological activity. Light sensing in both plant and certain animal-type cryptochromes has been shown to be triggered by flavin photoreduction to a stable radical intermediate form, which is involved in the signaling of the receptor [6], [7], [14]. Therefore, the ATP binding activity we have observed in vitro would be expected to increase the degree of activation and enhance the light responsivity of Atcry1 in vivo, thereby providing an additional mechanism to regulate this class of photoreceptor.

We have extended these observations on effect of ATP on cryptochrome function by showing that ATP binding by itself actually induces structural changes in the photoreceptor. These studies are particularly interesting in that conformational changes are induced by ATP binding already in the dark, without the requirement for light dependent electron transfer or subsequent flavin reduction to occur. Recently, the crystal structure of the PHR domain of Atcry1 has been reported to show no significant structural change upon binding to the non-hydrolysable AMP-PnP analog in prior studies [15]. However, the crystal structure was performed on a truncated N-terminal domain of Atcry1 and not the full-length protein. Since the C-terminal has been proposed to be the effector domain and thereby undergo profound conformational change during signaling reactions [17], [20], [21], it may be that ATP could lead to a strong conformational change that implicates the C-terminal domain of cryptochrome.

To date, higher plant (Arabidopsis cry1 and cry2), green algal Chlamydomonas cryptochrome and mammalian cryptochromes (Hscry1 and 2) have been reported to bind ATP [11], [12], [13], [14], [15]. Since these plant and animal-type cryptochromes are not directly evolutionarily related to each other, it is likely that regulation of cry function by ATP binding is another instance of convergent evolution in photoreceptor function. In this respect it is interesting that autokinase activity, that has also been reported in both plant and animal-type photoreceptors, does not seem to be required for enhanced photoreduction activity. Autokinase activity occurs in plant Atcry1, where it is light regulated [11], [12], in Chlamydomonas cryptochrome, where it is light regulated [14], and in Hscry1, where it is not reported to be light regulated [11], [13]. Apparent confusion on whether Atcry1 autokinase activity is light regulated is resolved by the different methodologies used in certain studies [11], [13]. There is as yet no clear evidence for biological function of cryptochrome autophosphorylation in vivo, although correlations were noted of blue light and phosphorylation with nuclear localization of Atcry2 or phosphorylation occurring subsequent to activation of Atcry1 [3], [12], [21], [22]. However, these studies involved phosphorylation reactions observed in vivo and may therefore be due to activity of heterologous plant kinases rather than resulting from autokinase activity, for which there is as yet no clearly defined biological role.

From the present study, it seems evident that ATP binding in and of itself may have a biological role and result in regulation of cryptochrome even in the absence of further autokinase activity. ATP binding alone appears to be sufficient to induce a conformational change in Atcry1, which, upon subsequent light activation, leads to enhanced photoreduction and formation of the signaling state of the photoreceptor. Thus, ATP binding could lead to enhanced light activation and biological activity of Atcry1 without additional autokinase activity. A further intriguing possibility is that ATP binding may affect the activity of the cryptochromes even in the absence of light. In plants, most known cryptochrome responses require activation by light and therefore this form of regulation is unlikely to occur. However, in the case of animal cryptochromes, the known roles of mammalian cryptochromes occur largely independently of light [3]. Therefore, a conformational change such as that induced by ATP binding could very well play a direct functional role mammalian cryptochromes without any requirement for light. Experiments to test this possibility are currently in progress.

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Acknowledgments 

We are indebted to Rachel Carol for critical reading of the manuscript and helpful discussion. This work was funded by the US National Science Foundation (Award No. 0343737) to Margaret Ahmad. Nathalie Hoang was funded by an ACI/BCMS doctoral fellowship from the French ministry of research.

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PII: S0014-5793(09)00229-4

doi:10.1016/j.febslet.2009.03.040

FEBS Letters
Volume 583, Issue 9 , Pages 1427-1433, 6 May 2009

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