View
217
Download
0
Category
Preview:
Citation preview
NATURE CHEMISTRY | www.nature.com/naturechemistry 1
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S1
Supplementary Information for
A protein engineered to bind uranyl selectively and with femtomolar affinity
Lu Zhou, Mike Bosscher, Salih Özçubukçu, Changsheng Zhang, Liang Zhang, Wen Zhang,
Charles J. Li, Jianzhao Liu, Mark P. Jensen, Luhua Lai*, Chuan He*
*To whom correspondence should be addressed. E-mail: chuanhe@uchicago.edu;
lhlai@pku.edu.cn.
Contents
Supplementary Methods…………………………………………………………….…………..2
Figure S1: Strategy for the development of uranyl-binding proteins…………………………...16
Figure S2: Uranyl coordination design and URANTEIN algorithm……………………….…...17
Figure S3: Scoring for evaluating coordination structure……………………………………….18
Figure S4: Uranyl-binding site at the interface of three monomers…………………………….20
Figure S5: Uranyl titration with protein and competitor………………………………………..21
Figure S6: Crystal structure comparison of SUP apo-form and complex with uranyl………….22
Figure S7: Details of the L67T interaction model and a structural comparison between SUP and
the original template protein…………………………………………………………………..23
Figure S8: Excess of MBP-SUP fusion immobilized on amylose resin can remove over 90%
uranyl in seawater. ..……………………..………………………………………………………24
Figure S9: Western blot of SUP-OmpA protein………………………………………………...25
Figure S10: The thermal stability assay of SUP showing the Tm to be ~71°C………………….26
Figure S11: Speciation diagrams for uranyl. ……………………………………………….......27
Figure S12: Gel filtration data for SUP with or without unrayl present………………………...28
Table S1: 40 uranyl coordination cases from PDB……………………………………………...29
Table S2: Detailed information of 10 designed uranyl-binding proteins………………………..30
Table S3: Uranyl-binding affinities of selected mutant proteins………………………………..32
Table S4: Selectivity of SUP to metal ions in seawater…………………………………………33
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 2
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S2
Table S5: Data collection and model statistics of SUP apo-form and SUP-uranyl complex…...34
Table S6: The primers used for the cloning and mutation of the synthesized gene…………….35
Table S7: Solution compositions and equilibrium constants used to determine the Kd of SUP by
carbonate competition and for speciation calculations at 25 °C. ……………………………….36
Table S8: Distribution of major uranyl-containing species in solution. ………………………..37
Supplemental References………………………………………………………………………38
Supplementary Methods and Equilibrium Modeling
Speciation calculations
Uranyl hydrolysis constants, stability constants for uranyl carbonate complexes,38,39 and
protonation constants of the carbonate/bicarbonate anions40 at the appropriate ionic strength
(Supplementary Table S7) were used to calculate the concentration of free uranyl in the
carbonate competition and sorption from freshwater experiments. Calculation of the speciation in
-log [H+] = 8.14 seawater (Supplementary Table S8) also included a correction to the free
carbonate concentration to account for ion pairing.41 Speciation curves are depicted in
Supplementary Figure S11. The resulting free uranyl concentration determined for seawater was
(2.4 ± 0.3) x 10-17 molal (2.4 x 10-17 M) at a total uranyl concentration of 1.39 x 10-8 molal (1.37
x 10-8 M).
If effective competition between carbonate and SUP for uranyl in seawater is defined as
the point where equal amounts of uranyl are carbonate-bound and SUP-bound (i.e., 6.85 x 10-9
M), then the two mass balance equations for uranyl and SUP can be combined with the
dissociation constant expression for UO2(SUP) to determine the Kd required to compete with
carbonate at a given SUP concentration and the appropriate uranyl concentrations for seawater,
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 3
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S3
Kd required = 2[SUP]total[UO22+]free ([UO2
2+]total – [UO22+]free)-1 – [UO2
2+]free
= 3.52 x 10-9 [SUP]total – 2.41 x 10-17 M
The maximum practical total SUP concentration for uranium recovery from seawater is
approximately 100 M. This corresponds to a Kd required of 350 fM or lower. Lower total SUP
concentrations would require still higher affinities, for example if [SUP]total = 10 M, the Kd
required would be 35 fM or lower. For the lowest possible SUP concentration that could bind
half of the total uranyl, 6.85 x 10-9 M, the Kd required is ~ 10-20 M.
The uranyl hydrolysis constants are well known for low ionic strength solutions or
solutions of NaClO4 electrolytes, but they are not well defined for NaCl or seawater solutions.38
Given this limitation, a series of speciation calculations to determine the impact of uranyl
hydrolysis on the calculated distribution of uranyl species was necessary. The infinite dilution
hydrolysis constants (Supplementary Table S7), which represent a reasonable maximum possible
formation of uranyl-hydroxide species in the NaCl or seawater systems, were used to test the
impact of uranyl hydroxides on the uranyl speciation in a series of model calculations. In these
model cases, which displayed the maximum possible fraction of uranyl-hydroxide species in
seawater or carbonate competition experiments, the calculations demonstrated that the uranyl
speciation was dominated by carbonate and protein complexes (Supplementary Table S8,
Supplementary Fig. S11). Any uranyl hydroxide species accounted for less than 0.1% of all the
uranyl species in the seawater systems, and no more than 2% of all uranyl species at the lowest
carbonate concentrations in the carbonate Kd determinations. Hydrolytic species had no
significant effect on the concentration of free uranyl cation (and thus the calculated Kd) in these
systems. However, the carbonate concentration in the pH 7 uranyl sorption from water
experiment was much lower because this experiment did not use added carbonate. In that case,
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 4
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S4
uranyl hydroxides were important species regardless of the uranyl concentration (Supplementary
Table S8). Fortunately, the hydrolysis constants are well known under those conditions (ionic
strength < 0.01 M).38 Similar model calculations were also used to test the importance of
UO2Cl+, (UO2)2CO3(OH)3-, (UO2)3(CO3)6
6-, (UO2)4(OH)7+, (UO2)3(OH)7
-, (UO2)2(OH)3+,
UO2(HPO4), UO2(OH)2 H2O (s), and UO2CO3 (s) using literature binding constants.38 None of
these species, alone or together, had a discernible impact on the distribution of dissolved uranyl-
carbonate or protein species or on the free uranyl concentration.
Generation of a library of potential binding residues
Generally, each residue was computationally mutated to aspartate, glutamate, asparagine, or
glutamine using the basic Mayer rotamer library42. If the mutated side-chain did not conflict with
surrounding residues, then the information regarding the oxygen position, linked carbon, and
corresponding rotamer was deposited to an oxygen library and, in the case of aspartate and
glutamate, a carboxyl library. The native aspartate and glutamate oxygens and main-chain
oxygens were also deposited in the appropriate library. To efficiently search for hydrogen bonds
between uranyl and the scaffold protein, the hydrogens of the native scaffold were collected to a
hydrogen library.
Screening using the URANTEIN algorithm
The URANTEIN algorithm searches for uranyl-binding sites in a scaffold protein that satisfies
one of the three coordination features introduced in Fig. S2c - S2e using a series of five steps in
Fig. S2f. First, a carboxyl is selected from the carboxyl library. The theoretical position of the
coordinated uranyl is calculated using standard coordination parameters: the O-U-O line is
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 5
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S5
perpendicular to the carboxyl plane, and the distances between the two carboxyl oxygens and the
uranium are equal at 2.46Å. If this uranyl is not in serious conflict with the surrounding residues,
the oxygens which can coordinate with this uranyl will be searched from the oxygen library in
step 2. Second, every element in the oxygen library was checked for the extent to which it
satisfied the coordination requirement. A coordination score is provided, which includes U-O
distance, C-O-U angle, planarity with the first carboxyl, repulsion with the first carboxyl, and
repulsion between the corresponding mutated residue and the uranyl (Fig. S3a). Next, the results
were filtered so that all remaining results had at least one carboxyl plus one oxygen from a
different residue without serious repulsion. Subsequently, if at least one coordination structure
was found, then hydrogen bonding was searched via the hydrogen library of the scaffold. Finally,
the uranyl binding sites were evaluated by a scoring function containing oxygen coordination,
oxygen compatibility, and hydrogen bonding (Fig. S3a).
Further Selection
From the top 5,000 hits we first narrowed down to 500-1000 hits of each three coordination
types by calculating the buried area of uranyl ion. Uranyl should be in a pocket with over 2/3
uranyl surface buried. The hit protein should also have entrance for uranyl binding. If uranyl is
completely buried in a protein core, the model was eliminated. We also deleted redundant results
with high sequence similarity in this step. Only the hit with the highest score among the
homologic results was retained. Secondly, we narrowed down to about 20 hits of each three
coordination types manually by considering the local environment of binding site, including
potential steric clashes and stability of coordination residues. Models with potential steric clashes
were eliminated. The coordination residues including mutated ones should locate on the
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 6
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S6
segments with secondary structure to make the binding motif rigid enough during the mutation
process. Accordingly, the results with better scores have higher probability to be selected.
Finally, we selected the 10 hits by evaluating the remaining results based on perceived thermal
stability (proteins from thermophilic organisms are preferred) and origin of organism. For
example, most human proteins are eliminated because they are less likely to express well in E.
coli, whereas U09, which consists of bundled α-helices, is chosen over comparable hits with
more random coil.
Plasmid construction and mutation
U01-U10 and SUP-OmpA fusion gene were synthesized by GeneScript. The gene sequences of
U09 and SUP-OmpA fusion protein are shown below. Mutations were performed using Pfu
Ultra II polymerase from Agilent. U01-U10 and all U09 mutations were cloned and expressed in
pMCSG19 vector for expression in BL21 or PRK1037 E. coli as previously described43. All
plasmid DNA was purified using a spin mini-prep kit, eluted into Type A water. The primers
used for the cloning and mutation of the synthesized gene are listed in Table S6.
U09 gene:
CTG GAT TGC CGT GAA CGC ATT GAA AAA GAC CTG GAA AAC CTG GAA AAA
GAA CTG ATG GAA ATG AAA AGC ATC AAA CTG TCT GAT GAC GAA GAA GCG
GTG GTT GAA CGT GCC CTG AAT TAT CGC GAT GAC AGT GTC TAT TAC CTG GAA
AAA GGC GAT CAT ATT ACC TCC TTT GGT TGT ATC ACG TAC GCG CAG GGC CTG
CTG GAT AGC CTG CGT ATG CTG CAC CGC ATT ATC GAA GGT
SUP-OmpA fusion gene:
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 7
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S7
ATG AAA GCT ACT AAA CTG GTA CTG GGC GCG GTA ATC CTG GGT TCT ACT CTG
CTG GCA GGT TGC TCC AGC AAC GCT AAA ATC GAT CAG GGA ATT AAC CCG TAT
GTT GGC TTT GAA ATG GGT TAC GAC TGG TTA GGT CGT ATG CCG TAC AAA GGC
CAG CGT GAA AAC GGT GCA TAC AAA GCT CAG GGC GTT CAA CTG ACC GCT
AAA CTG GGT TAC CCA ATC ACT GAC GAC CTG GAC ATC TAC ACT CGT CTG GGT
GGC ATG GTA TGG CGT GCA GAC ACT AAA TCC AAC GTT TAT GGT AAA AAC CAC
GAC ACC GGC GTT TCT CCG GTC TTC GCT GGC GGT GTT GAG TAC GCG ATC ACT
CCT GAA ATC GCT ACC CGT CTG GAA TAC CAG TGG ACC AAC AAC ATC GGT GAC
GCA CAC ACC ATC GGC ACT CGT CCG GAC AAC GGC GGA GGT TCT GGA GGA
GGG AGC AAT GCC CTG GAT TGC CGT GAA CGC ATT GAA AAA GAC CTG GAA
AAC CTG GAA AAA GAA CTG ATG GAA ATG AAA AGC ATC AAA CTG TCT GAT
GAC GAA GAA GCG GTG GTT GAA CGT GCC CTG AAT TAT CGC GAT GAC AGT
GTC TAT TAC CTG GAA AAA GGC GAT CAT ATT ACC TCC TTT GGT TGT ATC ACG
TAC GCG GAG GGC CTG ACG GAT AGC CTG CGT ATG CTG CAC CGC ATT ATC GAA
GGT
Expression and purification of protein
The strains carrying the plasmids were grown in LB to OD600 = 0.6, induced with 1 mM IPTG
and cells were grown overnight at room temperature before harvesting. Cells were lysed by
sonication in the presence of 1 mM PMSF as serine protease inhibitor. Supernatant was
separated by centrifugation and filtration through 0.45 μm PVDF. Ni-NTA columns were run
using 10 mM Tris pH 7.4, 500 mM NaCl with 1 mM DTT and imidizole ramping from 0 to 500
mM. Ni-NTA column chromatography gave pure protein in good yields. Protein samples were
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 8
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S8
concentrated using 10 kDa cutoff centrifuge filters and desalted into appropriate buffers. The
His-tag was removed by TEV protease. All crystallization samples were further purified by gel
filtration. Gel filtrations were run in 10 mM Tris pH 7.4 with 100 mM NaCl containing 1 mM
dithiothreitol (DTT).
Protein crystallization
Crystals of SUP apo-form and SUP-uranyl complex were originally identified using the PEG
ION crystallization screen kit (Hampton Research). Optimized crystals were produced using
hanging drop vapor diffusion at 16 °C by mixing 1 μL of protein solution at 10-20 mg/mL with 1
μL reservoir solution containing 2% v/v Tacsimate pH 4.0, 0.1 M sodium acetate trihydrate pH
4.6, 16% PEG 3350. For SUP-uranyl complex, uranyl was mixed with SUP at 1.2:1 molar ratio
before the drop set. Crystals appeared after 1 day and continued to grow for one week.
Crystal handling and data collection
Crystals were transferred to cryoprotectant consisting of a modified reservoir solution
supplemented with 15% glycerol (v/v) and quickly frozen in liquid nitrogen. All diffraction data
sets were collected at 100K at the macromolecular crystallography for life science beamline
LS/CA-CAT (21-ID-F) and NE-CAT (24-ID-C), respectively, at the Advanced Photon Source,
Argonne National Laboratory. Native data sets extending to 1.30 Å resolution were collected at
0.9795 Å wavelength (12.66 keV). Protein-uranyl complex data sets extending to 1.29 Å were
collected at the uranium L3-edge (17.18 keV, 0.7218 Å). The data were processed with
HKL200044 and the scaled data were used for molecular replacement. Crystallographic statistics
are summarized in Table S5.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 9
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S9
Data refinement
For phasing, model building and refinement, the structures of both UBP apo-form and UBP-
uranyl complex were determined by molecular replacement using Phaser in the CCP4 suite45,
with the template protein as the search model (pdb code: 2PMR). The structures were then
refined by using Phenix46. Manual rebuilding of the model was carried out using the molecular
graphics program COOT47 based on electron density interpretation. Water molecules were
incorporated into the model if they gave rise to peaks exceeding 3σ in Fo-Fc density maps. The
final refined model had good stereochemistry with 99.4% of the residues in the most favored
regions of the Ramachandran plot with none in the disallowed regions (Table S5).
Arsenazo III determination of uranyl
A modification of the Arsenazo III method48 was employed to determine uranyl concentrations
unless otherwise indicated. 50 μL of 80 μM Arsenazo III containing 0.1 M HCl was titrated with
an equal volume of uranyl solutions ranging from 0 to 30 μM, and the absorbances at 652 nm
and 800 nm were monitored. The value of A652-A800 increases linearly in this range, and can be
converted to uranyl concentrations. For high DGA (diglycolic acid) or carbonate concentrations
HCl was added to the final Arsenazo solution to compensate for the buffering activity of the
carboxylates.
Diglycolic acid competition assays
Diglycolic acid (DGA) competition assays were performed at pH 6.0 or 6.5 in 10 mM Bis-Tris
buffer with 300 mM NaCl.9 Standard solutions of 100 μM protein (expressed by PRK1037 E.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 10
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S10
coli with his-tag present, with an extinction coefficient of 7575 M-1 cm-1) and 100 μM UO22+
were prepared and diluted 10 fold with the appropriately scaled DGA buffer. The final solutions
contained 10 mM Bis-tris (pH 6.5), 10 μM protein, 10 μM UO22+ and different concentrations of
DGA. The solutions were mixed and filtered through 3 kDa cutoff centrifuge filters. Flow-
through was tested for uranyl concentration following Arsenazo III assay. The experiments were
repeated in triplicate.
Carbonate competition assays
Carbonate competition assays were performed similarly to the DGA assays, but with freshly
prepared pH 8.0-9.0 carbonate solutions in Tris-HCl buffer. All water used to prepare solutions
was freshly degassed and deionized and protected against further sorption of atmospheric CO2.
The final solution contained 10 mM Tris-HCl (pH 8.0-9.0), 10 μM protein, 10 μM UO22+ and
different concentrations of carbonate. Each binding curve was measured in three replicate
experiments. The reproducibility of each individual binding measurement at a given total
carbonate concentration was ±5%.
The Kd of the uranyl-protein complex was determined by fitting the resulting binding
curve using the uranyl-carbonate binding constants in Supplementary Table S7. (Inclusion of the
uranyl hydrolysis constants did not alter the calculated Kd in these experiment). The three
independent sets of carbonate competition data for each protein were combined and fit together
using two different approaches. First, the binding curve was fit by non-linear least squares
regression to the Hill equation implemented in the program Origin 8.5 (OriginLab), allowing the
minimum and maximum of the curve, the midpoint, and the Hill coefficient to vary
simultaneously. In the second approach, the Kd was calculated directly from the uranyl mass
balance equation at each experimental point, and the 15 individual Kd values between 10% and
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 11
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S11
95% protein binding were averaged. Each approach gave Kd values that were not statistically
distinguishable at the 95% confidence level. Nevertheless, the Kd values reported in Table S3
were based on the fit to the Hill equation, and the stated uncertainty incorporates both the
uncertainty in the total concentration of carbonate required for 50% binding derived from the fit
and the systematic uncertainty introduced by the uncertainties in the carbonate binding constants.
Resin immobilized U09 mutants
SUP protein (expressed by PRK1037 E. coli with his-tag present, an extinction coefficient of
7575 M-1 cm-1) was immobilized on Sulfhydryl Coupling Resin from G-Biosciences® (786-794)
by suspending the commercial slurry and washing both resin and protein with 50 mM Tris pH
8.5 coupling buffer containing 5 mM EDTA and 1 mM TCEP (tris(2-carboxyethyl)phosphine) to
generate free sulfhydryl. The resin and protein were combined with excess protein and held at
room temperature with mixing for 30 minutes. The reaction flow-through was collected to
determine efficiency and the resin was washed with the Tris coupling buffer. The resin was
quenched with 50 mM L-Cysteine HCl in coupling buffer mixed at room temperature for 30
minutes. Prior to use the resin was washed again with coupling buffer.
Metal competition assay
Metal competition assays of SUP were originally performed using protein immobilized on resin.
1 mL solutions of 500 nM uranyl with metals at different concentrations were prepared. The
solutions were mixed and incubated with resin in two aliquots for one minute each. The solution
was removed by vacuum filtration and the resin was washed thrice with 750 μL of 10 mM Bis-
Tris buffer at pH 6.5. We eluted with 50 μL of 20 mM carbonate solution at pH 9.0 in 10 mM
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 12
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S12
Tris. We used the Arsenazo III method48 to detect uranyl, and tested at a maximum ratio of 2.0
x 106 metal ions to 1 uranyl ion. If no uranyl was detected, we tested the competition again with
metal diluted ten-fold and repeated until uranyl was detected. After each test, the resin was
regenerated by rinsing with 750 μL saturated EDTA solution followed by 750 μL of 20 mM
carbonate solution at pH 9.0 in 10 mM Tris and washing with three aliquots of 750 μL of 10 mM
Bis-Tris buffer at pH 6.5.
To ensure that the presence of competing cations was not affecting the Arsenzo III assay, the
metal competition assay was repeated using amylose resin and ICP-MS for detection. SUP-MBP
fusion protein was immobilized on amylose resin at a concentration of 7 mg/mL. The assay
solutions contain 500 nM uranyl, different metals (based on the solubility, ranging from 1 mM to
2 M ) and 1 mM DGA (diglycolic acid) to prevent the precipitation and non-specific binding of
uranyl ion. The pH value of each mixed metal solution was measured. For each sample, 1 mL
mixed metal solution was incubated with 0.3 mL amylose resin for 1 minute. The supernatant
was diluted by 1% HCl and determined by ICP-MS.
Seawater sequestration assays by amylose resin
For preparation of SUP-bound amylose resin, SUP was cloned into pMCSG19 vector and
expressed in BL21 E. coli to produce an MBP fusion protein. Cells were harvested, sonicated,
and purified by nickel-NTA chromatography. The resulting protein was concentrated and
exchanged into a 10 mM Tris buffer at pH 7.4 containing 1 mM EDTA and 100 mM NaCl. The
solution was mixed by shaking and then exchanged to a 10 mM Tris buffer at pH 8.0 containing
100 mM NaCl. The concentration was measured by absorbance at 280 nm with an extinction
coefficient of 75290 M-1 cm-1. The protein was then incubated with amylose resin (New England
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 13
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S13
Biolabs, E8021S) in aliquots for 30 min. The supernatant was removed and the protein
concentration was tested. New protein aliquots were added until the resin was saturated. The
solution was washed with pH 8.0 Tris buffer containing no protein and the eluent was tested for
the presence of any protein. This procedure was repeated until no protein was eluted. The final
concentration was calculated at around 60 nmol per mL of concentrated resin bed volume. The
resin was then aliquoted as a wet slurry to 50 mL centrifuge tubes in amounts corresponding to
equimolar protein on resin and uranyl in seawater and 10:1 protein to uranyl. Larger resin
amounts were not tested due to the limits of eluting and testing from large resin samples. The
resin was incubated at room temperature in 50 mL of synthesized seawater under constant gentle
inversion for 30 minutes. The solution was then spun at 5,000 rpm for 10 minutes to concentrate
the resin at the bottom of the tube. The solution was then vacuum filtered through a QIAquick®
Spin Column (Mat. No. 1018215). In the last 5 mL of solution, the resin was resuspended by
pipetting gently and filtered with the solution. The flow-through was collected and analyzed by
ICP-MS. The resin was dried by spinning for 1 min at 8,000 rpm. The resin was then treated
with 20 mM carbonate solution at pH 9.2 for 30 min at room temperature in 100 μL aliquots.
The resin was centrifuged at 8,000 rpm for 1 min and the flow-through collected and analyzed by
Arsenazo III. If any uranyl was detected the resin was washed again with carbonate until no
more uranyl was detected. For the ion exchange approach, MBP-SUP fusion protein (60 mg)
was immobilized on 10 mL amylose resin in a ϕ10 mm column and pre-washed by non-uranyl
synthesized seawater. Total 10 mL seawater sample flowed through the column at a rate of 2
mL/min, and the flow-through was collected and analyzed by ICP-MS.
Seawater sequestration assays by surface-displayed cells
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 14
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S14
For preparation of surface-displayed cells, SUP-OmpA fusion protein was cloned into pBAD
vector and expressed in BL21 E. coli, with a linker GGGSGGGS between SUP and OmpA. Cells
were induced by 2% L-arabinose overnight and harvested by centrifugation at 6000 rpm. Cells
were washed with degassed, deionized H2O prior to use. For the seawater extraction assay, SUP-
bound amylose resin (5 mL) or surface display cells (0.5 mL pellet) were incubated with 50 mL
synthesized seawater for 30 minutes at room temperature. Supernatants were sent for uranium
analysis by ICP-MS. Blank resin or non-induced cells were used as negative controls and no
uranium enrichment was observed.
Western blot
The surface display cells were grown to 0.6 (OD600) and induced with and without 2% L-
arabinose overnight in LB. The bacteria were collected and suspended in buffer A (500 mM
NaCl, 10 mM Tris·HCl, pH 7.4, 1 mM DTT, 5% glycerol), followed by sonication to lyse the
bacteria. The 20-μL supernatants were loaded into a 12% SDS page gel for separation. After
standard Western blot procedures, the proteins were detected by anti-FLAG antibody (Sigma-
Aldrich, A8592).
Thermal stability assay
Fresh SUP protein purified by Ni-NTA column and gel filtration column was applied in the
thermal shift assay study. The experiment was performed on Applied Biosystems® 7500 Fast
Real-time PCR system using sypro-orange as the fluorescence dye and followed the standard
procedure of protein thermal shift studies provided by Applied Biosystems. Briefly, SUP protein
(0.25 µg/µL, final concentration) was mixed with dye (5-10X, final concentration) in total 20 µL
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 15
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S15
solution (50 mM Bis-tris, pH 6.0, 50-400 mM NaCl) and heated from 10 to 95 ℃ at 1% heating
rate. The fluorescence signals were monitored using the ROX (Ex:584 nm, Em: 612 nm) as
reporter. The data were analyzed by Protein Thermal ShiftTM software (Invitrogen, version 1.1).
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 16
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S16
Supplementary Figure S1. Strategy for the development of uranyl-binding proteins. In our
strategy for the development of uranyl-binding proteins binding motifs were identified and the
protein database was screened for appropriate binding models. Virtual hits were expressed,
tested, and crystallized. Crystal and experimental data were used to develop new mutants which
were again tested, crystallized in mutant form. This loop continued until satisfactory binding
efficiencies were reached or the scaffold no longer yielded improved mutants.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 17
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S17
Supplementary Figure S2. Uranyl coordination design and URANTEIN algorithm. The
crystal structure of uranyl nitrate hexahydrate (5) is the reference for protein-binding-uranyl
coordination design(a and b). Uranyl is represented by blue and red ball and stick model. In this
structure, six oxygens from four nitrate oxygens and two water oxygens coordinate the uranium
atom in the equatorial plane. The distance of U-nitrate O is 2.50 Å, and U-water O is 2.4 Å. In
our design, nitrate was replaced with the carboxyl of an aspartate or glutamate side-chain, and
one or two waters were replaced with the carbonyl oxygen of an asparagine or glutamine side-
chain, main-chain carbonyl oxygen, or monodetate coordinate aspartate or glutamate (c).
Conformations with two adjacent carboxyl ligands (d), or three carboxyl ligands (e) were also
considered.(f) The URANTEIN algorithm uses a five-step screening strategy based on preferred
uranyl coordination geometries.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 18
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S18
Supplementary Figure S3. a, Scoring for evaluating coordination structure.
1) Oxygen coordination requirement and evaluation.
i
i parameter of coefficentOscore (Oscore should be greater than 0.4)
Parameter coefficent
U-O(Å) 1.8-2.2: 0.5 2.2-2.8: 1.0 2.8-3.2: 0.8 3.2-3.6: 0.4
∠COU(º) 60-85: 0.5 85-180: 1.0
min(∠OUOA,
∠OUOB) (º) 60-75: 0.7 75-90: 1.0
min(O-O1, O- 1.9-2.3: 0.4 2.3-2.6: 0.8 >2.6: 1.0
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 19
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S19
O2) (Å)
R repulsion If R is not carboxyl or min(∠O’UOA, ∠O’UOB) < 60º, then all
atoms of R cannot repulse with UO2 and the first carboxyl
2) Hydrogen bonding requirement and evaluation.
i
i parameter of coefficentHscore (Hscore should be greater than 0.4)
Parameter coefficient
OA-H(Å) 1.5-1.8: 0.5 1.8-2.5: 1.0 2.5-3.0: 0.8 3.0-3.6: 0.4
∠OAHroot (º) 60-90: 0.4 90-120: 0.7 120-180: 1.0
∠UOAH (º) 60-90: 0.4 90-120: 0.7 120-180: 1.0
3) Oxygen-oxygen compatible requirement and evaluation.
ij
jii
of postion residue theat not ist coefficien compatible O-O compatO (should be greater than 0.4)
4)
Uranyl coordination structure score.
j
j
i
ii scoreHscoreOcompat)O1icienttype_coeffUscore (
b, Crystal structure of 2PMR, a scalfold of U09 selected for further optimization. SUP were
designed based on the mutation of 2PMR, which was a protein of unknown function from
Methanobacterium thermoautotrophicum.
c, Designed coordination environment.
The predicted environment of U09 with the mutations identified in the URANTEIN screening
algorithm shows two bidentate and two monodentate equatorial ligands as well as a hydrogen
bond for one of the axial oxo-ligands.
Compatible coefficient
O1-O2(Å) 1.9-2.3: 0.4 2.3-2.6: 0.8 >2.6: 1.0
Main-chain Wild carboxyl Else wild Mutate
carboxyl else
Type_coefficient 2.0 1.8 1.5 1.2 1.0
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 20
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S20
Supplementary Figure S4. Uranyl-binding site at the interface of three monomers. a, The
trimer interface binding site is overviewed b, and detailed and shows an idealized uranyl-binding
motif featuring 5 equatorial coordinates by three glutamate and one water molecule. A hydrogen
bond is also observed between the uranyl axial oxo-ligand and side chain asparagine39.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 21
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S21
Supplementary Figure S5. a) Uranyl binding affinity of SUP with DGA. Competition assay
of SUP vs. DGA for uranyl yields a Kd of 0.2±0.1 nM at pH 6.0 in 300 mM NaCl. b) Uranyl
titration with protein and competitor. A stock solution of 10 μM protein and 10 mM DGA
was prepared in Bis-tris buffer (pH 6.0) with 300 mM NaCl. Uranyl was added to aliquots of the
stock to create solutions ranging from 0 to 30 uM. At low uranyl concentrations, most of the
uranyl (see panel a) is bound to the protein in the designated binding site (Kd = 0.2 nM at pH 6.0
and 7.4 fM at pH 8.87) The sharp increase in eluted uranyl above 12 μM total uranyl indicates
saturation of the primary binding site and that there are no other specific interactions between
uranyl and the protein with a Kd lower than 4 nM.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 22
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S22
Supplementary Figure S6. Crystal structure comparison of SUP apo-form and complex
with uranyl. Arg71 forms a hydrogen bond with an axial oxo of the uranyl ion in the complex
structure (green) but has different conformation in apo-form (magenta). Glu17 also has a
conformational change in apo-form compared to the SUP-uranyl complex structure, showing that
uranyl binding is site specific.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 23
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S23
Supplementary Figure S7. Details of the L67T interaction model and a structural
comparison between SUP and the original template protein. a, Thr67 forms a hydrogen bond
with Ala64 main chain (shown in stick) near the uranyl-binding pocket by Glu17, Asp68 and
Arg71 (shown in line). The newly formed hydrogen bond stablizes the helix structure, thus
increasing the binding affinity. b, Structural comparison between SUP (green) and template
protein 2PMR (blue). Residue 67 (Thr in SUP and Leu in 2PMR) are shown in stick. The last
helix bent in SUP compared to template protein, showing that hydrophobic core residue (Leu67)
mutation significantly alters the conformation.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 24
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S24
Seawater Flow through0
1
2
3
4
5SeawaterFlow through
U c
once
ntra
tion
(ppb
)
Supplementary Figure S8. Excess of MBP-SUP fusion immobilized on amylose resin can
remove over 90% uranyl in seawater. MBP-SUP fusion protein (60 mg) was immobilized on
10 mL amylose resin in a 10 mm column and pre-washed by non-uranyl synthesized seawater. A
total of 10 mL seawater sample flowed through the column at a rate of 2 mL/min, and uranyl
concentration of the flow-through was analyzed by ICP-MS.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 25
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S25
Supplementary Figure S9. Western blot of SUP-OmpA protein. Western blot showing that
SUP-OmpA fusion protein can be effectively expressed after L-arabinose induction. The original
E.coli OmpA (~19 kD) was replaced by overexpressed fusion protein (~ 26 kD).
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 26
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S26
Supplementary Figure S10. The thermal stability assay of SUP protein. a) the protein
showed the Tm to be ~71 °C in the buffer containing 50 mM Bis-tris, pH 6.0, 50 mM NaCl. b)
the protein’s Tm did not alter under different salt concentration.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 27
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S27
Supplementary Figure S11. Speciation diagrams for uranyl. The speciation for (a) 13 nM
uranyl in seawater with Kd UO2(SUP) = 7.4 fM and (b) 667 nM uranyl in pH 7.0 water that is in
equilibrium with atmospheric CO2 with Kd UO2(SUP) = 0.2 nM. Calculations were performed
using the program Medusa52 and the equilibrium constants and solution compositions given in
Supplementary Table S7. Solid squares (■) represent the amount of uranium sorbed to the SUP
resin at (a) 1:1 10:1 and 6100:1 SUP:U ratios in seawater or (b) a 30:1 SUP:U ratio in water.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 28
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S28
Supplementary Figure S12. Gel filtration data for SUP with or without unrayl present. The
peaks of SUP were shown at the same position with 50 µM uranyl present (a) or without uranyl
(b) in a Superdex 200 column (GE healthcare). The buffer contains 20 mM Tris-HCl and 100
mM NaCl.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 29
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S29
Supplementary Table S1. 40 uranyl coordination cases from PDB.
PDB Uranyl Coordination oxygen U-O (Å) ∠COU 1BZO IUM A 502 OD1 ASP A 76 2.716 91.847 1BZO IUM A 502 OD2 ASP A 76 2.622 94.320 1BZO IUM A 559 OE1 GLU A 53 2.628 99.995 1BZO IUM A 559 OE2 GLU A 53 2.846 87.477 1CT9 IUM A1101 OD1 ASP A 238 2.022 146.092 1CT9 IUM A1101 OD1 ASP A 351 2.378 138.977 1CT9 IUM A1102 OE1 GLU A 352 2.841 126.868 1CT9 IUM A1102 OD1 ASP A 384 2.555 130.752 1CT9 IUM A1103 OE1 GLU A 352 3.034 136.543 1CT9 IUM A1103 OD2 ASP A 384 2.626 105.627 1EFQ IUM A 199 OD1 ASP A 38 2.406 96.439 1EFQ IUM A 199 OD2 ASP A 38 2.629 86.087 1EFQ IUM A 199 O GLN A 42 2.593 163.335 1FE4 IUM A1003 OD2 ASP A 32 2.486 129.421 1JET IUM C 1 OD2 ASP A 323 2.293 129.306 1JET IUM C 4 OE2 GLU A 251 2.296 154.126 1JET IUM C 4 OD2 ASP A 369 2.230 129.646 1JET IUM C 5 OD2 ASP A 362 2.274 130.063 1JET IUM C 5 OD2 ASP A 410 2.430 127.318 1JET IUM C 8 OD2 ASP A 11 2.621 143.065 1JET IUM C 9 OE1 GLN A 220 2.267 154.747 1NCI IUM A 300 OE2 GLU A 11 2.523 136.444 1NCI IUM A 300 OD1 ASP A 67 2.547 129.806 1T9H IUM A 402 OE1 GLU A 105 2.594 89.664 1T9H IUM A 402 OE2 GLU A 105 2.441 96.968 1T9H IUM A 407 OD1 ASP A 108 2.432 97.966 1T9H IUM A 407 OD2 ASP A 108 2.497 95.023 2VEO IUM A1441 OD2 ASP A 292 2.112 175.153 2VEO IUM A1441 OE1 GLU A 298 2.758 94.309 2VEO IUM A1441 OE2 GLU A 298 2.991 83.706 2VEO IUM A1442 OD2 ASP A 220 2.465 129.713 2VEO IUM A1442 OE2 GLU A 314 2.165 154.291 3L0O IUM A 428 OD1 ASP A 77 2.868 94.248 3L0O IUM A 428 OD2 ASP A 77 2.877 93.786 3L0O IUM A 428 OD1 ASP A 84 2.845 100.686 3L0O IUM A 428 OD2 ASP A 84 3.071 89.829 3L0O IUM A 430 OE1 GLU A 14 2.546 100.933 3L0O IUM A 430 OE2 GLU A 14 2.812 87.916 3L0O IUM B 429 OE1 GLU B 219 3.007 97.383 3L0O IUM B 429 OD2 ASP B 222 2.889 129.778 3L0O IUM B 431 OD2 ASP B 77 2.799 119.021
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 30
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S30
Supplementary Table S2. Detailed information of 10 designed uranyl-binding proteins.
Data for the top ten uranyl-binding protein candidates identified by the in silico screen. The
mutation column indicates mutations necessary for the native protein to fulfill the screen
conditions. The residues column indicates the residues expected to bind uranyl in the equatorial
positions.
EXPNO.
PDB Code Protein name Mutatio
n Sequence (after mutation) Coordination residues
U01 2G9M
pigment protein phycoerythrin from Cyanobacterium
Asn121Glu Pro123Asp
QRAAARLEAAEKLGSNHEAV VKEAGDACFSKYGYNKNPGE AGENQEKINKCYRDIDHYMR LINYTLVVGGTGPLDEWGIA GAREVYRTLELDSAAYIAAF VFTRDRLCAPRDMSAQAGVE FCTALDYLINSLS
Glu071 Glu121 Asp123
U02 2O6F rTp34 from
Treponema pallidum
Asp074Gln Ala119Gln
DEFPIGEDRDVGPLHVGGVY FQPVEMHPAPGAQPSKEEAD CHIEAQIHANEAGKDLGYGV GDFVPYLRVVAFLQKHGSEK VQKVMFAPMNQGDGPHYGAN VKFEEGLGTYKVRFEIAAPS HDEYSLHIDEQTGVSGRFWS EPLVAEWDDFEWKGPQW
Glu072 Gln074 Gln119 Asp121
U03 1QR4
Fibronectin type-III domain from chicken tenascin
Val145Glu Val133Glu
GSTVVGSPKGISFSDITENS ATVSWTPPRSRVDSYRVSYV PITGGTPNVETVDGSKTRTK LEKLVPGVDYNVNIISVKGF EESEPISGILKTALDS
Glu133 Lys143 Glu145
U04 1JRL E. coli
Lysophospholiase L1
Leu011Asp Gly072Glu Ile156Asp
ADTLLILGDSDSAGYRMSAS AAWPALLNDKWQSKTSVVNA SISGDTSQQGLARLPALLKQ HQPRWVLVELGENDGLRGFQ PQQTEQTLRQILQDVKAANA EPLLMQIRPPANYGRRYNEA FSAIYPKLAKEFDVPLLPFF MEEVYLKPQWMQDDGDHPNR DAQPFIADWMAKQLQPLVNH DSLE
Asp011 Glu072 Asp156
U 1T0A 2C-Methyl-D- Ile111Gl MKIRIGHGFDVHKFGEPRPL Asp058
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 31
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S31
05 Erythritol-2,4-cyclodiphosphate Synthase from Shewanella Oneidensis
u Lys061Asn
ILCGVEVPYETGLVAHSDGD VVLHAISDAILGAMALGDIG NHFPDTDAAYKGADSRVLLR HCYALAKAKGFELGNLDVTI IAQAPKMAPHEEDMRQVLAA DLNADVADINVKATTTEKLG FTGRKEGIAVEAVVLLSRQ
Asn061 Glu111
U06 2J80
Periplasmic domain of sensor histidine kinase CITA
His009Glu Leu106Asp Ile129Glu Leu132Ala
MDITEERLEYQVGQRALIQA MQISAMPELVEAVQKRDLAR IKALIDPMRSFSDATYITVG DASGQRLYHVNPDEIGKSME GGDSDEALINAKSYVSVRKG SLGSSDRGKSPIQDATGKVI GIVSVGYTEEQAE
Glu009 Asp106 Glu129
U07 3HDP
Ni(II)-bound Glyoxalase-I from Clostridium acetobutylicum
Cys077Glu Ala104Glu His5Thr Glu124Ala
GSHMSLKVHTIGYAVKNIDS ALKKFKRLGYVEESEVVRDE VRKVYIQFVINGGYRVELVA PDGEDSPINKTIKKGSTPYH IEYEVEDIQKSIEEMSQIGY TLFKKAEIEPAIDNRKVAFL FSTDIGLIALLEK
Glu052 Glu077 Glu104
U08 1IIU
Chicken plasma retinol-binding protein (RBP)
Lys017Glu
MDCRVSSFKVKENFDENRYS GTWYAMAKKDPEGLFLQDNV VAQFTVDENGQMSATAKGRV RLFNNWDVCADMIGSFTDTE DPAKFKMKYWGVASFLQKGN DDHWVVDTDYDTYALHYSCR ELNEDGTCADSYSFVFSRDP KGLPPEAQKIVRQRQIDLCL DRKYRVIVHNGFCS
Glu017 Gly051 Asp079
U09 2PMR
unknown function from Methanobacterium thermoautotrophicum
Asn017Glu Leu013Asn His064Gln
LDCRERIEKDLENLEKELME MKSIKLSDDEEAVVERALNY RDDSVYYLEKGDHITSFGCI TYAQGLLDSLRMLHRIIEG
Asn013 Glu017 Gln064 Asp068
U10 2FA5
multiple antibiotic-resistance repressor (MarR) from Xanthomonas campestris
Ser027Glu Ile049Asp Arg053Asp
MSDLDTPTPSPHPVLLNLEQ FLPYRLEVLSNRISGNIAKV YGDRYGMADPEWDVITILAL YPGSSASEVSDRTAMDKVAV SRAVARLLERGFIRRETHGD DRRRSMLALSPAGRQVYETV APLVNEMEQRLMSVFSAEEQ QTLERLIDRLAKDGLPRMASKD
Asp049 Asp053 Glu027
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 32
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S32
Supplementary Table S3. Uranyl-binding affinities of selected mutant proteins.
Uranyl-binding efficiencies for 4 top virtual hits and for optimized mutants of U09. The binding
affinity of first generation hits are around the 10-7 to 10-8 M level. Each successive mutation to
U09 yielded significant increases in binding affinity.The data were repeated at least three times.
Mutant Dissociation constant (Kd)
U02 98±25 nMa U04 98±7 nMa U09 37±9 nMa 56±1 pMb U10 92±38 nMa
U09 Leu67Thr 1.8±0.3 nMa 5.0±5pMb U09Gln64Glu, Leu67Thr 1.0±0.3 pMb, 20±8 pMa,
SUP (U09Asn13Asp, Gln64Glu, Leu67Thr) 7.4±2.0 fMc, 0.2±0.1 nMd,
a pH 6.5, DGA assay b pH 8.15, carbonate assay c pH 8.87, carbonate assay d pH 6.0, DGA assay
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 33
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S33
Supplementary Table S4. Selectivity of SUP to metal ions.
Seawater levels of seventeen metals are expressed as concentrations and molar excess compared
with uranyl in column 1-3. These values are given as a comparison to the measured selectivity
of SUP for uranyl against each metal. The conditions and results of competition experiment
evaluated by ICP-MS were listed in column 4-6, including pH value of metal-uranyl mixture,
metal concentration and uranyl concentration in the supernatant. The selectivity was calculated
based on the uranyl concentration in the supernatant and molar excess of metal ions.
Metal [Seawater] Excess pH Metal
concentration
(M)
Uranyl concentrat
ion (incubated
with amylose
resin, nM)
Selectivity
Blank resin
6.0 284±6
Na+ 470 mM 3.5*107 4.7 2 82±13 9.8±1.6* 106 Mg2+ 53 mM 3.9*106 4.4 2 154±20 3.4±0.4 * 106 K+ 10 mM 7.6*105 6.1 2 108±13 6.5±0.8 * 106
Ca2+ 10 mM 7.7*105 5.5 1 147±12 1.9±0.2 * 106 Sr2+ 8.9 uM 660 4.0 1 155±2 1.7±0.02 * 106 Rb+ 1.4 uM 110 8.2 1 141±1 2.0±0.01 * 106 Ba2+ 110 nM 8.1 5.1 1 43±2 1.1 ±0.05*107 VO2+ 39 nM 2.9 2.9 0.01 193±9 9.4±0.4*103 Pb2+ 13 nM 1 3.3 0.1 36±3 1.4±0.1*105 Ni2+ 8.2 nM 0.63 4.0 1 61±1 7.3±0.1 * 106 Zn2+ 5.4 nM 0.42 <2 1 ND 2 *106 Cu2+ 2.4 nM 0.18 5.1 0.001 105±2 3.4±0.06*103 Hg2+ 700 pM 0.054 3.4 0.1 71±1 6.0±0.08*105 Cd2+ 620 pM 0.048 5.6 1 55±3 8.3±0.4 *106 Fe3+ 540 pM 0.041 <2 1 ND 2 * 106 Mn2+ 360 pM 0.028 6.0 1 221±5 5.7±0.1 *105 Co2+ 20 pM 0.0015 5.6 1 38±2 1.3±0.07 *107
ND: not determined because of protein precipitation in the metal solution.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 34
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S34
Supplementary Table S5. Data collection and model statistics of SUP apo-form and SUP-
uranyl complex.
SUP-apo SUP-uranyl Data Collection Space group P212121 P212121 Cell dimensions a, b, c (Ǻ) 44.26, 47.03, 65.44 44.03, 46.93, 65.76 α, β, γ (°) 90.00 90.00 90.00 90.00 90.00 90.00 Resolution* (Ǻ) 40-1.30 (1.35-1.30) 40-1.29 (1.34-1.29) Completeness (%) 98.2 (89.2) 97.4 (92.0) Rmerge 0.032 (0.071) 0.054 (0.616) I/σI 36.3 (14.0) 15.7 (1.6) Redundancy 4.0 (2.3) 2.0 (1.9) Refinement Resolution* (Ǻ) 38.2-1.30 (1.33-1.30) 38.2-1.29 (1.31-1.29) Completeness (%) 98 (85) 97 (87) No. reflections 33856 64466 (34552) Rwork/Rfree 16.0 (17.6) 17.9 (18.8) Complex in asymmetry unit 2 2 Protein residues 82 82 Ligands 0 2 Most favoured# (%) 99.4 99.4 Additionally allowed (%) 0.6 0.6 Disallowed (%) 0 0 B-factor Protein : 10.6
Solvent: 24.9 Protein : 15.7 Solvent: 28.9 Uranyl: 15.6
R.m.s. deviations Bond lengths (Ǻ) 0.007 0.007 Bond angles (°) 1.094 1.034 PDB accession code 4FZO 4FZP
* Highest resolution shell is shown in parenthesis.
# Values calculated using PROCHECK from CCP4 suite.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 35
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S35
Supplementary Table S6. The primers used for the cloning and mutation of the synthesized
gene.
Name Sequence Purpose U09_MCSG_F U09_MCSG_R
TACTTCCAATCCAATGCCCTGGATTGCCGTGAACGCATTGAAAAA TTATCCACTTCCAATGTTAACCTTCGATAATGCGGTGCAGCATA
MCSG Cloning
U09_L67T_F U09_L67T_R
GTACGCGCAGGGCCTGACGGATAGCCTGCGTATG CATACGCAGGCTATCCGTCAGGCCCTGCGCGTAC
Mutagenesis
U09_Q64E_F U09_Q64E_R
ATCACGTACGCGGAGGGCCTGACGG CCGTCAGGCCCTCCGCGTACGTGAT
Mutagenesis
U09_E17A_F U09_E17A_R
CCTGGAAAACCTGGAAAAAGCACTGATGGAAATGAAAAGC GCTTTTCATTTCCATCAGTGCTTTTTCCAGGTTTTCCAGG
Mutagenesis
U09_E17Q_F U09_E17Q_R
CCTGGAAAACCTGGAAAAACAACTGATGGAAATGAAAAGC GCTTTTCATTTCCATCAGTTGTTTTTCCAGGTTTTCCAGG
Mutagenesis
U09_D68A_F U09_D68A_R
GAGGGCCTGACGGCTAGCCTGCGTATG CATACGCAGGCTAGCCGTCAGGCCCTC
Mutagenesis
U09_D68N_F U09_D68N_R
GGAGGGCCTGACGAATAGCCTGCGTATGC GCATACGCAGGCTATTCGTCAGGCCCTCC
Mutagenesis
U09_N13D_F U09_N13D_R
TGAACGCATTGAAAAAGACCTGGAAGACCTGGAAAAAGAAC GTTCTTTTTCCAGGTCTTCCAGGTCTTTTTCAATGCGTTCA
Mutagenesis
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 36
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S36
Supplementary Table S7. Solution compositions and equilibrium constants used to
determine the Kd of SUP by carbonate competition and for speciation calculations at 25 °C.
Carbonate binding constants are adjusted for different ionic strengths using specific ion
interaction coefficients38. The components used in the equilibrium expressions are H+, UO22+,
Ca2+, CO32-, DGA2- and SUP.
Kd determination
Sorption of UO22+
from water Sorption of UO2
2+ from seawater
Ionic Strength, molal 0.30 < 0.01 0.72 [CO3
2-]free , molal 0 – 0.0110 2.15 x 10-8 1.98 x 10-5 a [UO2
2+]total , molal 1.0 x 10-5 6.66 x 10-7 1.30 x 10-8 -log [H+] 8.02 / 8.74 b 7.00 8.14 c
Species log i or log hydr H2O (pKw) 13.72 14.00 13.22 CO2 (aq) ↔ CO2 (g) 1.49 1.47 1.53 HCO3
- 9.65 10.33 8.91 H2CO3
15.67 16.68 14.75 UO2(CO3) 8.84 9.94 8.72 UO2(CO3)2
2- 15.48 16.61 15.35 UO2(CO3)3
4- 21.91 21.84 22.00 Ca2UO2(CO3)3 n.c. d n.c. d 26.89 UO2(OH)+ -5.25 e -5.25 -5.25 e UO2(OH)2 -12.15 e -12.15 -12.15 e UO2(OH)3
- -20.55 e -20.55 -20.55 e UO2(OH)4
2- -32.4 e -32.4 -32.4 e (UO2)2(OH)2
2+ -5.62 e -5.62 -5.62 e (UO2)3(OH)5
+ -15.55 e -15.55 -15.55 e HDGA- 2.82 f H2DGA 6.58 f UO2(DGA) 5.11 f UO2(DGA)2
2- 7.54 f a Total [CO3
2-] concentration in seawater calculated from constants in Appendix A of ref. 40 and corrected for ion pairing after ref. 41 b Proton activity coefficient from ref. 49, –log[H+] = 8.02 for U09Gln64Glu, Leu67Thr protein , –log[H+] = 8.74 for SUP protein. c Proton activity coefficient from ref. 50 d Species not considered in calculations because these solutions lack Ca e Maximum estimated value of the hydrolysis constant at this ionic strength. f pH = 6.0. Equilibrium constants from ref. 51 and proton activity coefficient from ref. 49.
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 37
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S37
Supplementary Table S8. Distribution of major uranyl-containing species in solution.
Calculated for various solution compositions, in the absence of protein, from the equilibrium
constants in Supplementary Table S7.
Sorption of UO22+
from water Sorption of UO2
2+ from water
Sorption of UO22+
from seawater Ionic Strength, molal < 0.01 < 0.01 0.72 [CO3
2-]free , molal 2.15 x 10-8 2.15 x 10-8 1.98 x 10-5 a [UO2
2+]total , molal 6.66 x 10-7 b 1.26 x 10-8 c 1.30 x 10-8 -log [H+] 7.00 7.00 8.14 d
Species Fraction total uranyl UO2
2+ 0.002 0.003 1.8 x 10-9
UO2(OH)+ 0.114 0.165 UO2(OH)2 (aq) 0.143 0.208 UO2(OH)3
- 0.011 0.017 (UO2)3(OH)5
+ 0.311 <0.001 UO2(CO3) (aq) 0.379 0.511 UO2(CO3)2
2- 0.038 0.062 0.002 UO2(CO3)3
4- 0.137 Ca2UO2(CO3)3 0.861 a Total [CO3
2-] concentration in seawater calculated from constants in Appendix A of ref. 40 and corrected for ion pairing after ref. 41 b 158 ppb uranium c 3 ppb uranium d Proton activity coefficient from ref. 50
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 38
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S38
Supplementary References and Notes
38. Guillaumont R. et al., Eds., Update on the chemical thermodynamics of uranium, neptunium,
plutonium, americium, and technetium, vol. 5 (Elsevier, New York, 2003).
39. Kalmykov, S. N., Choppin, G. R. Mixed Ca2+/UO22+/CO3
2- complex formation at different ionic
strengths. Radiochim. Acta 88, 603-606 (2000).
40. Zeebe, R. E., Wolf-Gladrow, D. CO2 in Seawater: Equilibrium, Kinetics, Isotopes, volume 65 of
Elsevier Oceanography Series. Elsevier, New York (2001).
41. Pytkowicz, R. M., Hawley, J. E. Bicarbonate and carbonate ion-pairs and a model of seawater at
25°C. Limnol. Oceanogr. 19, 223-234 (1974).
42. Maeyer, M. D., Desmet, J., Lastres, I. All in one: a highly detailed rotamer library improves both
accuracy and speed in the modelling of sidechains by dead-end elimination. Fold Des. 2, 53-66
(1997).
43. Donnelly, M. I., Zhou, M., Millard, C. S., Clancy, S., Stols, L., Eschenfeldt, W. H., Collart, F. R.,
Joachimiak, A. An expression vector tailored for large-scale, high-throughput purification of
recombinant proteins. Protein Expression Purif. 47, 446-454 (2006).
44. Otwinowski, W. Z. Minor Processing of X-ray diffraction data collected in oscillation mode. Methods
Enzymol. 276, 307 (1997).
45. Read, R. J. Pushing the boundaries of molecular replacement with maximum likelihood. Acta
Crystallogr. D Biol. Crystallogr. 57, 1373-1382 (2001).
46. Adams, P. D., Afonine, P.V., Bunkóczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung,
L.W., Kapral, G.J., Grosse-Kunstleve, R.W., McCoy, A.J., Moriarty, N.W., Oeffner, R., Read, R.J.,
Richardson, D.C., Richardson, J.S., Terwilliger, T.C., Zwart, P.H., PHENIX: a comprehensive
Python-based system for macromolecular structure solution. Acta Cryst. D 66, 213-221 (2010).
47. Emsley, P.; Cowtan, K. Statistical phase improvement without a solvent boundary. Acta Crystallogr.
D Biol. Crystallogr. 60, 2126-2132 (2004).
© 2014 Macmillan Publishers Limited. All rights reserved.
NATURE CHEMISTRY | www.nature.com/naturechemistry 39
SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.1856
S39
48. Rohwer, H., Rheeder, N., Hosten, E. Interactions of uranium and thorium with Arsenazo III in an
aqueous medium. Anal. Chim. Acta 341, 263-268 (1997).
49. Harned, H. S., Owen, B. B. The Physical Chemistry of Eectrolytic Solutions, volume 137 of ACS
Monograph Series. Reinhold: New York (1958).
50. Khoo, K. H., Ramette, R. W., Culberson, C. H., Bates, R. G. Determination of hydrogen ion
concentrations in seawater from 5 to 40 °C: Standard potentials at salinities from 20 to 45‰. Anal.
Chem. 49, 29-34 (1977).
51. Smith, R., Martell, A., Motekaitis, R. NIST Critically Selected Stability Constants of Metal
Complexes Database vol. 8; Standard Reference Data Program. NIST, Gaithersburg, MD (2004).
52. Puigdomenech, I. MEDUSA: Make Equilibrium Diagrams Using Sophisticated Algorithms, Royal
Institute of Technology (KTH), Stockholm (2004). http://www.kth.se/medusa.
© 2014 Macmillan Publishers Limited. All rights reserved.
Recommended