Supplemental Data. Engelhard et al. Plant Cell (2014) 10.1105/tpc.114.129809
Δ Absorbance
C
Δ Absorbance
B
D
Δ Absorbance
Δ Absorbance
A
Supplemental Figure 1. W374A mutant photoreduction in whole cell lysates after the addition of
NADH, NADPH, and ATP.
Whole cell lysates of the W374A mutant was dialyzed overnight and illuminated with
400 µmol m-2 s-1 white light (WL) for 3 minutes. Light versus dark difference spectra were
obtained in the absence (dotted line) or presence (solid line) of added 1 mM NADH, NADPH,
ATP or all three as indicated. No added thiol reductant was used.
Supplemental Data. Engelhard et al. Plant Cell (2014) 10.1105/tpc.114.129809
A.
B.
DARK
LIGHT
1 µM NADH
10 µM NADH
100 µM NADH
0.5 mMNADH
1.0 mMNADH
2.0 mMNADH
Absorbance (A.U.)
DARK
LIGHT
0.001 mMATP
0.01 mMATP
0.1 mMATP
0.5 mMATP
1.0 mMATP
400
500
600
Wavelength (nm)
400
450
500
550
600
Wavelength (nm)
Supplemental Figure 2. Photoreduction of isolated W374A protein at different metabolite
concentrations.
Isolated proteins at a concentration of 10 mM were illuminated for 3 min in white light (WL,
400 µmol m-2 s-1) in the presence of 10 mM β-mercaptoethanol and increasing concentrations
of ATP (panel A) or NADH (panel B). Threshold and saturation was at around 10 mM
concentration for ATP (panel A) whereas threshold for NADH was at 10 mM with saturation
at 0.5 mM (panel B).
Supplemental Data. Engelhard et al. Plant Cell (2014) 10.1105/tpc.114.129809
% binding
A
B
cry2 Y399A
cry2
0.5
0
cry1
0.5
1
1
0.5
0.5
0
0
0
20
200
40
µM ATP
400
60
0
0
0
20
200
40
400
60
µM ATP
C
cry2
w e
Y399A Y399F
w e w e
Supplemental Figure 3. ATP binding affinity in wild type and mutant cryptochromes.
Binding affinities were determined for wild type cry2 and Y399A isolated mutant proteins
(panel A) and for cry1 as a control (panel B). Kd determination was performed as described in
Supplemental Methods, error bars represent S.D. of three independent measurements. Data is
presented as the percent ATP bound as a function of ATP concentration. Inset – shows values
at higher ATP concentrations (to 500 mM) to demonstrate saturation. Kd values are as
follows: cry2 = 49 +- 12 mM, Y399A = 12 +- 2 mM, and cry1 = 23 +- 8 mM. Panel C shows
binding of isolated cry2 wild type, Y399A, and Y399F proteins to ATP agarose beads. The
isolated proteins were applied to ATP agarose beads and eluted from the column with 1 mM
ATP as described (see Supplemental Methods). An additional final wash was performed at the
same volume as the subsequent elution step. The presence of cry2 protein in the eluted
fraction (indicated by 'e') as well as in the equivalent concentration of the final wash prior to
elution (indicated by ‘w’) was determined by western blot analysis with anti-cry2 antibody.
The position of the cry2 proteins is indicated by the arrow, and is specifically eluted by ATP.
Supplemental Data. Engelhard et al. Plant Cell (2014) 10.1105/tpc.114.129809
Supplemetal Figure 4. Absorbance changes in E. coli DNA photolyase upon UV light
illumination in the presence of different reductants.
E. coli DNA photolyase (25 µM) was illuminated with LEDs (385 nm; 270 µmol m-2 s-1,
15°C) and spectra were recorded via Maya spectrometer for 30 min. Reductants used were 1
mM dithiothreitol (DTT) (A), 1 mM NADPH (B), 1 mM NADH (C) or 1 mM glutathione
(D). Kinetics were plotted for the decrease in 445 nm absorbance of fully oxidized FAD (E)
and the data fitted by mono-exponential decay curves. Half-lives of the 445 nm absorbing
Supplemental Data. Engelhard et al. Plant Cell (2014) 10.1105/tpc.114.129809
Absorbance (A.U.)
flavin species under different reductant-conditions were calculated (F).
DARK
LIGHT
NAD
NADP
400
500
600
Wavelength (nm)
Supplemental Figure 5. Photoreduction of isolated W374A protein in the presence of NAD+
and NADP+.
Isolated proteins were illuminated for 3 minutes in white light (WL, 400 µmol m-2 s-1) in the
presence of 10 mM β-mercaptoethanol. NAD+ and NADP+ were added at a concentration of
1 mM.
Supplemental Data. Engelhard et al. Plant Cell (2014) 10.1105/tpc.114.129809
Supplemental Figure 6. cry2 is eluted from an ATP agarose affinity column by added
NADH.
Isolated cry2 protein was applied to ATP agarose beads and eluted from the column with
either 5 mM NADH or 5 mM ATP as described (see Supplemental Methods). The presence of
cry2 protein in the eluted fraction (indicated as 'elution') as well as in the final wash prior to
elution was determined by Western blot analysis with anti-cry2 antibody. A control
experiment was performed with agarose beads that were not crosslinked to ATP (indicated as
'agarose') as an additional verification of cry2 binding specificity.
Supplemental Data. Engelhard et al. Plant Cell (2014) 10.1105/tpc.114.129809
Supplemental Figure 7. Partial proteolysis of cry2 in the presence or absence of ATP.
Isolated cry2 proteins were incubated with trypsin (see Supplemental Methods). Lane
designated 'T0' represents intact full-length cry2 protein prior to addition of protease. Protein
concentration in all lanes was identical with the exception of the T0 sample (which was at 5fold dilution). Sample aliquots were removed from 5 to 60 min subsequent to addition of
trypsin protease and resolved on SDS gel. Cleavage of a fragment of about 17 kDa occurs
within 5 min and is identical in both samples (compare 5' point to T0). However, samples
with added ATP were protected from further proteolysis as compared to control samples. In
particular, two proteolytic cleavage fragments (A and B) indicated by arrows accumulate in
control (minus ATP) samples over a period of at least 40 min that are absent in the presence
of ATP. Experiments gave similar results over three replicative trials. Differential proteolysis
is indicative of conformational change in cryptochromes (Partch et al., 2005; Kondoh et al.,
2011).
Absorbance
Supplemental Data. Engelhard et al. Plant Cell (2014) 10.1105/tpc.114.129809
0.06
T0
WL
0.04
WL+ATP
0.02
0.00
400
500
600
Wavelength (nm)
Supplemental Figure 8. ATP binding facilitates cry2 photoreduction at low pH.
Isolated cry2 protein was adjusted to pH 5.7 in 50 mM phosphate buffer, 10 mM βmercaptoethanol. A spectrum was taken before illumination (T0) and again after 10 seconds
illumination in white light (400 µmol m-2 s-1) in the absence (WL) or presence (WL + ATP) of
1 mM added ATP. pH of the sample was measured before and after illumination, to verify
that no changes occurred.
Supplemental Data. Engelhard et al. Plant Cell (2014) 10.1105/tpc.114.129809
SUPPLEMENTAL METHODS
ATP-agarose affinity chromatography
Binding assays were performed as follows: After two washes with 1 ml PBS buffer (50 mM
NaH2PO4, 150 mM NaCl, pH 7.5), 0.1 ml of adenosine 5′-triphosphate immobilized on cross
linked 4% beaded agarose (Sigma, cat. no. A2767) were incubated with 2 µg of purified cry2
in PBS for 2 h at 4°C. After incubation, the beads together with bound protein were washed
five times with 1 ml PBS. The bound cryptochrome was eluted by incubation with 100 µl
PBS containing variously 5 mM ATP or 5 mM NADH for 1 h at 4°C. The eluted supernatant
was analyzed by SDS/PAGE, followed by Western blot analysis with anti-cry2 antibody using
standard methods (Bouly et al., 2007). An equivalent volume of the final wash prior to elution
(designated as 'wash' in the figure) was also analyzed. Binding reactions to agarose beads
without crosslinked ATP (designated 'Agarose' in Supplemetal Figure 4) were also performed
in parallel.
Partial proteolysis of cry2
For trypsin digestion kinetics equal quantities of full-length cry2 (10 µM) were digested in
PBS buffer and 0.05 ng/µl of trypsin at 20°C in the dark. Samples with 1 mM of ATP were
preincubated at 4°C with ATP for 5 min before addition of trypsin. The reactions were
stopped at indicated times with 5X SDS buffer, resolved by 12.5% SDS–PAGE and
visualized by Coomassie staining.
Photoreduction of E. coli photolyase
Expression and purification of E. coli DNA photolyase was performed under red light
conditions as described (Pokorny et al., 2005; Moldt et al., 2009). However, the sizeexclusion chromatography step was omitted, and as final buffer 50 mM Tris-HCl, pH 7.5, 100
mM NaCl, 40 % (v/v) glycerol was used. The protein was concentrated using Amicon ultra
centrifugal filter concentrators with a 30 kDa cutoff (Millipore). Protein samples with fully
oxidized flavin were prepared by a treatment of the protein with a 15 molar excess of
potassium ferricyanide (Moldt et al. 2009). The protein was then exchanged into fresh buffer
using several steps on Amicon ultra centrifugal filter concentrators with a 30 kDa cutoff
(Millipore).
Chemicals ß-nicotinamide adenine dinucleotide phosphate reduced form tetrasodium salt
(NADPH) and L-glutathione reduced form were purchased from AppliChem and βnicotinamide adenine dinucleotide reduced disodium salt (NADH) from Roth. Reducing agent
stock solutions were prepared immediately before usage.
Spectroscopic Studies
Absorption spectra were recorded using a DH-2000-BAL light source (Mikropack) and a
Maya 2000 Pro spectrometer (Ocean Optics). For photoreduction, the protein in 50 mM TrisHCl, pH 7.5, 100 mM NaCl, 10 % (v/v) glycerol and 1 mM of the different reducing agents
was incubated for 10 min in the dark and the first spectrum at t = 0 min was taken afterwards.
Subsequently, the sample was continuously illuminated with a high power LED at 385 nm
(270 µmol m-2 s-1, Roithner Lasertechnik) at 15°C. Absorbance spectra were recorded every
minute with an integration time of 13 ms and three scans for mean value over 30 min under
continuous UV light illumination. To determine half-lives of oxidized flavin in E. coli DNA
photolyase, absorbance at 445 nm during photoreduction was plotted and the data were fitted
Supplemental Data. Engelhard et al. Plant Cell (2014) 10.1105/tpc.114.129809
by mono-exponential decay curves.
Quantification of ATP binding (Kd determination).
Determination of binding constant was performed essentially as described (Bouly et al.,
2003). Briefly, cry protein (7 mg) was bound to a Ni2+ resin and subjected to stringent washes
as described for the purification procedure. Instead of eluting the bound protein, samples were
subsequently incubated for 2 h at 20°C in 50 mM HEPES (pH 7.0), 20 mM MgCl2 and
protease inhibitors with increasing concentrations of ATP containing [a-32P]ATP at constant
specific activity. After five washes with buffer containing 50 mM Tris/HCl (pH 7.5), 150 mM
NaCl and 10 mM imidazole, the radioactive ATP (cpm) bound to the immobilized cry1 was
counted. For Kd determination, the data was fitted in OriginPro using a one-binding-site
model A[cpm] = Amax * cATP[µM] / ( Kd[µM] + cATP[µM] ) [cpm].
Supplemental Data. Engelhard et al. Plant Cell (2014) 10.1105/tpc.114.129809
Supplemental References
Bouly, J. P., Giovani, B., Djamei, A., Mueller, M., Zeugner, A., Dudkin, E.A.,
Batschauer, A., and Ahmad M. (2003). Novel ATP-binding and autophosphorylation
activity associated with Arabidopsis and human cryptochrome-1. Eur. J. Biochem. 270:
2921-2928.
Bouly, J.P., Schleicher, E., Dionisio-Sese, M., Vandenbussche, F., Van Der Straeten, D.,
Bakrim, N., Meier, S., Batschauer, A., Galland, P., Bittl, R., and Ahmad, M. (2007).
Cryptochrome blue light photoreceptors are activated through interconversion of flavin redox
states. J. Biol. Chem. 282: 9383-9391.
Kondoh, M., Shiraishi, C., Müller, P., Ahmad, M., Hitomi, K., Getzoff, E.D., and
Terazima, M. (2011). Light-induced conformational changes in full-length Arabidopsis
thaliana cryptochrome. J. Mol. Biol. 413: 128-137.
Moldt, J., Pokorny, R., Orth, C., Linne, U., Geisselbrech,t Y., Marahiel, M.A., Essen,
L.O., and Batschauer, A. (2009). Photoreduction of the folate cofactor in members of the
photolyase family. J. Biol. Chem. 284: 21670–21683.
Partch, C.L., Clarkson, M.W., Ozgür, S., Lee, A.L., and Sancar, A. (2005). Role of
structural plasticity in signal transduction by the cryptochrome blue-light photoreceptor.
Biochemistry 44: 3795-3805.
Pokorny, R., Klar, T., Essen, L.O., and Batschauer, A. (2005). Crystallization and
preliminary X-ray analysis of cryptochrome 3 from Arabidopsis thaliana. Acta Crystallogr.
Sect. F Struct. Biol. Cryst. Commun. 61: 935–938.