S
1
SUPP
ORTING
INFORMATION
Characterization of the DNA
-
mediated Oxidation of Dps, a Bacterial Ferritin
Anna R. Arnold, Andy
Zhou, and Jacqueline K. Barton
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
California 91125, USA
Primer Sequences
Mutation
Primer
W52A
5
ʹ
-
GATCTTTCTTTGATTACCAAACAAGCGCAC
GCG
AACATGC
-
3
ʹ
(Forward)
5
ʹ
-
GCATGTT
CGC
GTGCGCTTGTTTGGTAATCAAAGAAAGATC
-
3
ʹ
(Reverse)
W52Y
5
ʹ
-
CAGTTTATTGATCTTTCTTTGATTACCAAACAAGCGCAC
TAC
AACATGC
-
3
ʹ
(Forward)
5
ʹ
-
GCATGTT
GTA
GTGCGCTTGTTTGGTAATCAAAGAAAGATCAA
TAAACTG
-
3
ʹ
(Reverse)
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2
Overexpression and purification of
E. coli
Dps
WT and
mutants
E. coli
Dps
proteins
were purified according to published procedures
(
1,2
)
with a number of modifications. A single colony of
E. coli
cell line ZK2473, which
consists of ZK2471 (
dps
::
kan
Δ
recA
-
(Cam)
Δ
ara
714, leu::Tn10) donated by Dr. Roberto Kolter
(
3
)
with the
pBAD18
-
dps plasmid, was used to inoculate 50 mL of LB media containing 100
μ
g/mL ampicillin and 50
μ
g/mL kanamycin and was grown overnight at 37°C with 200 rpm
shaking. The subsequent day,
5
mL of the overnight culture was used to inoculate 1 L cultures
o
f LB containing the same concentrations of antibiotics, which were grown at 37ºC with 150 rpm
shaking until OD
600
= 1. Overexpression of Dps was then induced with 0.3%
w/v
L
-
arabinose.
Af
ter 4 hours of incubation at 37
ºC with continued shaking, the cells
were harvested by
centrifugation for 10 minutes at 7,000
g
.
Cells were resuspended in Buffer A (50 mM Tris, pH 8, 500 mM NaCl, 0.1 mM EDTA)
containing a protease inhibitor cocktail (Roche) and lysed via microfluidization. The cell lysate
was clarified
by centrifugation at 15,000
g
for 15 minutes, followed by a second round of
centrifugation of the supernatant at 27,000
g
for 20 minutes.
The resulting supernatant was
fractionated by ammonium sulfate (AS) precipitation by slow addition of solid AS up to
60%
saturated AS, centrifugation for 15 minutes at 10,000
g
, and collection of the supernatant. Next,
more solid AS was added to the supernatant up to 90% saturated AS in order to precipitate Dps.
The
Dps protein
was recovered by centrifugation for 20 minutes at 27,000
g
, and the pellet
resuspended in approximately 5 mL of Buffer A. The partially
-
purified Dps was then dialyzed
against a low ionic strength buffer (20 mM Tris, pH 8), where it precipitated while col
ored
contaminants remained in solution. Precipitated protein was isolated by centrifugation at 26,000
S
3
g
for 10 minutes, and resuspended in Buffer B (
50 mM Tris, pH 8, 2 M NaCl, 0.1 mM EDTA).
The high salt concentration results in Dps dissociation from en
dogenous DNA.
Dps was separated from DNA and other contaminants by loading onto a HiLoad 16/600
Superdex 200 pg size exclusion column (GE Healthcare), and running with Buffer B. Fractions
were pooled that had an A
260
:A
280
ratio of 0.57, indicating
no n
ucleic acid contamination. E
xcess
salt was removed by
loading pooled fractions onto a
HiPrep 26/10 des
alting column (GE
Healthcare) equilibrated with Buffer C (50 mM Tris, pH 7
, 150 mM NaCl
). Next, the
partially
purified
Dps solution
was loaded
onto
a Hi
Trap
Heparin
HP
column (5 mL, GE Healthcare)
using
a
peristaltic pump
that had previously been equilibrated with Buffer C. The column was
then w
ash
ed
with 3 column volumes (CV)
of Buffer C
,
and Dps
elute
d
with a
n increasing
gradient of Buffer D (
50 mM Tri
s
, pH 7
, 1 M NaCl
) over 10 CV. Dps elutes at approximately
200 mM NaCl.
Finally,
purified
Dps
was exchanged into storage buffer
(50 mM Tris, pH 7, 150
mM NaCl, 20% glycerol) using a HiPrep
26/10 desalting column (GE Healthcare). From 4 L of
cell culture, the yield of purified Dps is approximately 130 mg. Protein purity was confirmed by
SDS
-
PAGE (data not shown).
References:
1
. Almiron, M.; Link, A.J.; Furlong, D.; Kolter, R.
Genes Dev
.
1
992
,
6
, 2646
-
2654.
2. Zhao, G.; Ceci, P.; Ilari, A.; Giangiacomo, L.; Laue, T.M.; Chiancone, E.; Chasteen, N.D.
J. Biol. Chem
.
2002
,
277
, 27689
-
27696.
3. Martinez, A.; Kolter, R.
J. Bacteriol.
1997
,
179
, 5188
-
5194.
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4
Figure
S1
. Wide EPR spectra of irradiated sample containing WT ferrous iron
-
loaded Dps,
poly(dGdC)
2
DNA,
Ru(phen)(dppz)(bpy
ʹ′
)]
2+
,
and
[Co(NH
3
)
5
Cl]
2+
with dark control subtracted.
All conditions and instrument settings identical to Figure
3 in manuscript
.
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5
Figure
S2
.
Ferrous iron only (no protein) mixed with stoichiometric ferricyanide in 50 mM Tris,
pH 7.0, 150 mM NaCl, 5% glycerol. Concentration of iron was 240
μ
M, equivalent to that in 20
μ
M
solution of Dps with 12 Fe/Dps. Instrument settings identical to Figure
3 in manuscript
.
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6
Figure
S3
.
WT Dps EPR spectra compared to Fe only control in MOPS buffer.
Concentrations:
Fe only: 240
μ
M
ferrous iron with 240
μ
M
ferricyanide (black trace). Irradiated Dps sample:
20
μ
M Dps (Fe
2+
/Dps: 11.
7
± 0.
1
), 1 mM base
-
pairs poly(dGdC)
2
DNA, 20
μ
M
[Ru(phen)(dppz)(bpy
ʹ′
)]
2+
, 120
μ
M [Co(NH
3
)
5
Cl]
2+
(red trace)
. Buffer: 50 mM
MOPS
, pH 7.0,
150 mM NaCl
, 5% glycerol. Dps spectrum
has had DC subtracted.
Instrument settings:
modulation amplitude = 10 G at 100 kHz; frequency = 9.37 GHz; microwave power =
6.4 mW
;
t
emperature = 10 K.