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Supplemental Methods
Protein Purification
T. weissflogii was grown as previously described1 in 25L polycarbonate carboys bubbled
with sterile filtered air. Cells from 300L of culture were harvested by filtration. Cells
from a 2L culture with 20mCi/L 109Cd added to the media were harvested by filtration
and added to the cell pellet as a tracer. The pellet was resuspended in lysis buffer (50mM
Tris-HCL pH 7.0, 1mM EDTA, 0.1mM DTT and 0.1% phenylmethyl sulfonyl fluoride)
and lysed by sonication on ice. Progress of cell lysis was followed by microscopy. The
crude cell lysate was centrifuged for 30 minutes at 10,000 rpm in a Beckman JA-10 rotor
at 4oC to remove frustules and other cell debris. This low speed supernatant was then
transferred and centrifuged at 50,000 rpm in a Beckman 60ti rotor for 2h at 4oC to pellet
membrane fragments. This high speed supernatant was subjected to sequential 30% and
70% ammonium sulfate precipitations. The pellet from the 70% ammonium sulfate
precipitation was resuspended in 200mL of 25mM sodium phosphate (pH 6.8) and 0.5M
ammonium sulfate (HICA buffer). 100mL was loaded at 2mL/min onto a 20mL butyl
hydrophobic interaction chromatography (HIC) column and washed with 5 column
volumes of HICA buffer at 5mL/min. Protein was eluted with a 10 column volume, 0.5 to
0M gradient of ammonium sulfate in 25mM phosphate buffer (pH 6.8) run at 2.5mL/min.
Forty 5mL fractions were collected. The HIC column was run twice and the carbonic
anhydrase activity in collected fractions was detected as previously described2. Briefly,
equal volumes of each fraction were run on 10% nondenaturing gels, soaked in 0.1%
bromthymol blue in 1x Lammeli running buffer (without SDS)3. Saturated CO2 gas
blown over the gel revealed yellow bands of CA activity. Gels were then dried and
radiolabeled bands were imaged by phosphorimaging. Pooled CA containing fractions
were concentrated by ultrafiltration and exchanged into 10mM histidine buffer (pH 6.0)
using a Biorad 10DG column. The protein mixture was then injected onto an HPLC with
a TSK-DEAE ion-exchange column and eluted with a NaCl gradient. Fractions were
assayed for CA activity and 109Cd label as described above, and fractions containing the
enzyme were pooled. Purified protein was assayed for enzyme activity, purity
(Coomassie stained) and 109Cd label using non-denaturing gels. N-terminal sequencing
was performed by automated Edman degradation at the Princeton University Core
Facility. Internal peptides were generated by trypsin digestion of the purified Cd-CA by
the method of Fernandez and Mische (1996)4. Peptides were purified by C-18 capillary
HPLC and sequenced as above. The peptide sequence of the amino terminus was
NQSNTSSSTSKASLTPDQIVAALQERGWQAIVTE FSLLN and that of the internal
peptide was IVIPSISPAQGAEL.
Sequence determination: PCR, cloning, sequencing
T. weissflogii total RNA was extracted from 400 mL of mid log phase culture using TriReagent (Sigma, St Louis MO). cDNA was synthesized using the Clontech Smart RACE
kit following the manufacturer’s instructions. The nearly full-length cDNA was cloned
in three steps (see Supplemental Figure 1). Degenerate primers P65-6 (sequence: 5'GGITGGCARACIGARATHG-3’) and P65-3b (5'-ARYTCIGCICCYTGIGCIGG-3')
were designed using the amino terminal and internal peptide sequences. PCR reactions
carried out with these primers yielded a 620 base pair product that contained both primer
sequences and also encoded the amino-terminal peptide sequence downstream of the
region targeted by P65-6 and upstream of the region targeted by P65-3b. Thus it was
clear that we had amplified and cloned a cDNA fragment that encodes a large portion of
the amino-terminal region of the Cd CA. Two nested non-degenerate primers CdCT-2
(5'- GGTCGACGTCGATCCTCAAGGC-3') and CdCT- 1 (5'CATCTTGAAATGCGTCCACGGACG-3') were then used sequentially in conjunction
with Clontech primers UP and NUP to amplify the last two thirds of the cDNA encoding
the CDCA1. Because the Cd-CA takes the form of three direct repeats of the 200 amino
acids each, the cDNA fragment encoding the amino terminus that was cloned in the first
step did not overlap the larger fragment encoding the carboxy terminus that was cloned in
the second step. Two separate fragments were cloned in spite of the fact that the primers
used for the amplification of the second fragment were designed using the sequence from
the first fragment. In the final step, to amplify the nearly full length cDNA encoding all
three direct repeats in the CDCA1 sequence, nested PCR was performed first using
primers CCA-4 (5'-CGGAATTCTCCCTCCTCAACG-3') and CCA-3 (5'CAAAAACTTGACCACATCCAA-3)'. The resulting product was diluted 1:10000 fold
and a nested PCR reaction was run using primers CCA-4 and CCA-2 5'CGACATCGTCGAGGCCTTGAC-3' and Clontech hot-start Advantage Polymerase.
The final PCR product was gel purified and cloned into a non-expression plasmid
(TOPO-TA Invitrogen). An additional clone of the entire CDCA1 gene was generated
using mutagenic PCR primers 5'-CTCCCTCCTCAACGAAATGGT-3' (CCA-070302A)
and 5'-CCCCGTCACAGCCATCATCTAAGG-3'(CCA-070302B) and its sequence was
determined as described above. The cdca1 sequence has been submitted to Genbank
(accession #AY772014).
X-ray Absorption Spectroscopy
Spectra were measured at the Stanford Synchrotron Radiation Laboratory (SSRL) on
beamline 7-3, using a Si(220) double crystal monochromator, with the SPEAR storage
ring containing 70-100 mA at 3.0GeV. Harmonics were rejected by detuning one
monochromator crystal to 60% of peak intensity, and samples were maintained at 10K in
an Oxford Instruments helium flow cryostat. Spectra were measured using a 30-element
Ge array detector, and incident and transmitted x-ray intensity was measured using Arfilled ionization chambers. X-ray energy was calibrated with reference to the lowest
energy inflection of a Cd metal foil, assumed to be 26714.0 eV. CdCA1 protein, purified
from T. weissflogii as described above, was used for analyses. Because the sample was
dilute (~ 7 M Cd) a total of 59 scans, each taking 25min, were averaged in order to
obtain adequate signal to noise.
References
1.
2.
3.
4.
Lane, T. W. & Morel, F. M. M. Proc. Natl. Acad. Sci. 97, 4627-4631 (2000).
Roberts, S., Lane, T. & Morel, F. Journal of Phycology 33, 845-850 (1997).
Sambrook, J., Fritsch, E. F. & Maniatis, S. Molecular Cloning: A Laboratory
Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York,
1989).
Fernandez, J. and Mishe S.M. The protein protocols handbook. J.M. Walker, ed.,
p 405-414, Humana Press, Totowa, NJ (1996).
Supplementary Figure 1. a.) The derived amino acid sequence of CDCA1:
three repeats are located within CDCA1 sequenced from T. weissflogii b.)
Alignment of the three repeats (R1-R3, where * indicates where the sequence
continues at the beginning of the subsequent repeat) of CDCA1 from
Thalassiosira weissflogii, and that derived from the entire homologous gene from
the genome of the marine diatom Thalassiosira pseudonana (Tp-CdCA).
a.
NQSNTSSSTSKASLTPDQIVAALQERGWQAEIVTEFSLLNEMVDVDPQGILKCVDGRGSDNTQFCGPKMPGG
IYAIAHNRGVTTLEGLKQITKEVASKGHVPSVHGDHSSDMLGCGFFKLWVTGRFDDMGYPRPQFDADQGAKA
VENAGGVIEMHHGSHAEKVVYINLVENKTLEPDEDDQRFIVDGWAAGKFGLDVPKFLIAAAATVEMLGGPKK
AKIVIPSISPAQIAEALQGRGWDAEIVTDASMAGQLVDVRPEGILKCVDGRGSDNTIMGGPKMPGGIYAIAH
NRGVTSIEGLKQITKEVASKGHLPSVHGDHSSDMLGCGFFKLWVTGRFDDMGYPRPQFDADQDANAVKDAGG
IIEMHHGSHTEKVVYINLLANKTLEPNENDQRFIVDGWAADKFGLDVPKFLIAAAATVEMLGGPKNAKIVVP
SITPPQIVSALRGRGWKASIVKASTMSSELKRVDPQGILKCVDGRGSDNTQFGGPKMPGGIYAIAHNRGVTT
LEGLKDITREVASKGHVPSVHGDHSSDMLGCGFFKLWLTGRFDDMGYPRPEFDADQGALAVRAAGGVIEMHH
GSHEEKVVYINLVSGMTLEPNEHDQRFIVDGWAASKFGLDVVKFLVAAAATVEMLGGPKKAKIVIP*
b.