A protein engineered to bind uranyl selectively andwith femtomolar affinityLu Zhou1†, Mike Bosscher1†, Changsheng Zhang2,3†, Salih Ozcubukcu1, Liang Zhang1, Wen Zhang1,
Charles J. Li1, Jianzhao Liu1, Mark P. Jensen4, Luhua Lai2,3* and Chuan He1*
Uranyl (UO221), the predominant aerobic form of uranium, is present in the ocean at a concentration of ∼3.2 parts
per 109 (13.7 nM); however, the successful enrichment of uranyl from this vast resource has been limited by the highconcentrations of metal ions of similar size and charge, which makes it difficult to design a binding motif that is selectivefor uranyl. Here we report the design and rational development of a uranyl-binding protein using a computationalscreening process in the initial search for potential uranyl-binding sites. The engineered protein is thermally stable andoffers very high affinity and selectivity for uranyl with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity overother metal ions. We also demonstrated that the uranyl-binding protein can repeatedly sequester 30–60% of the uranyl insynthetic sea water. The chemical strategy employed here may be applied to engineer other selective metal-bindingproteins for biotechnology and remediation applications.
Uranium is the key element for nuclear-energy production andis important in many other applications. The most stable andrelevant uranium ion in aerobic environments is the uranyl
cation, UO22þ. As a consequence of its solubility the concentration
of uranyl in sea water is surprisingly high at 3.2 mg per tonne of seawater (13.7 nM)1. Based on this concentration the ocean is esti-mated to contain 1,000 times more uranium than land contains,and so offers an enormous resource that, unlike land resources,may be tapped at minimal environmental cost.
Functionalized polymers with amidoxime-type ligands havebeen used to sequester uranyl in sea water since the 1980s2,3.However, the low binding affinity and selectivity of the ligandsused limited the utility of the approach. Other approaches basedon functional groups or small-molecule chelates have been pro-posed, with limited success4,5. Sea water is slightly basic and con-tains �2.2 mM total carbonate, which chelates uranyl stronglyand leaves a free uranium concentration of only 2 × 10217 M(Supplementary Tables 1 and 2, and Supplementary Fig. 1). Tocompete with this high concentration of dissolved carbonateunder practical conditions, a ligand that can bind uranyl withclose to femtomolar (fM, 10215) affinity is required (seeSupplementary Information). In addition, calcium(II) is present at10 mM in sea water and prefers an oxygen-rich environmentsimilar to that which typically binds uranyl. Therefore, a dauntingselectivity of 106-fold for uranyl over calcium is desired.Sophisticated chelating ligands6–11 have been developed for otheruseful applications, but high synthetic costs limit their utilizationwhen dealing with vast amounts of sea water. A layered solid-stateion exchanger, K2MnSn2S6, has been shown to have high affinityfor uranium in addition to many other metals12. Very recently,metal–organic frameworks (MOFs) have also been employed asnovel sorbents to extract uranyl from aqueous media13. Biologicalsystems, which can be self-regenerated, offer an opportunity toachieve economically both the required affinity and high selectivity.
Through billions of years of evolution nature has producedstrategies to recognize beneficial or toxic metal ions with highsensitivity and selectivity. In many cases, with the help of a well-folded protein scaffold and the assistance of second-sphereinteractions, metal ions can be recognized in the femtomolar tozeptomolar (10221) range with extremely high selectivity14–16. If acorrect and robust scaffold can be developed for the metal of inter-est, such affinities are sufficient for economic mining of uranium orother elements from sea water or to remediate polluted environ-mental sites. Additionally, proteins may be displayed on the surfacesof living organisms17, thus allowing for the biological regenerationof these systems for recovery and/or remediation purposes at verylow costs.
Uranyl-binding motifs are well known and offer distinctivehandholds for the rational design of proteins that selectively binduranyl7,8. Uranium prefers to oxidize to the þ6 state with twoaxial oxo ligands and form the linear triatomic uranyl ion withan overall charge of þ2. The ability to afford five or six equatorialligands in pentagonal or hexagonal bipyramidal geometry separatesuranyl from most of the alkali, alkaline and transition metals. Thepresence of the axial oxo ligands as potential hydrogen-bondacceptors also distinguishes uranyl from most known lanthanideand actinide species in the environment7. Although some uranyl-binding motifs have been designed in organic ligands, in DNAand in proteins6–11, none have been able to cross the affinitythreshold to compete with carbonate (2.2 mM in ocean) andachieve the selectivity requirements over other metals in seawater. Our strategy was to use computational screening anddesign to develop a stable protein with sufficient binding affinityfor uranyl and selectivity over other metals. The protein can beimmobilized on a solid support or displayed on the cell surfacefor repeated use and sequestration (Fig. 1). This approach can beapplied to both engineered systems (resin immobilization) andbiological systems (cell-surface display).
1Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA, 2BNLMS,State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering and Center for QuantitativeBiology, Peking University, Beijing 100871, China, 3Center for Life Sciences, Peking University, Beijing 100871, China, 4Chemical Sciences and EngineeringDivision, Argonne National Laboratory, Argonne, Illinois 60439, USA, †These authors contributed equally to this work. *e-mail: [email protected];[email protected]
ARTICLESPUBLISHED ONLINE: 26 JANUARY 2014 | DOI: 10.1038/NCHEM.1856
ResultsComputational screening for potential uranyl-binding sites. Wedeveloped a large-scale computational screening algorithm,named URANTEIN, to search the Protein Data Bank (PDB) forpockets that may accommodate uranyl (Fig. 2). A total of 12,173protein structures in the PDB were considered as scaffolds, onwhich every residue could be mutated to Asp, Glu, Asn or Glnusing the corresponding rotamers in silico. Based on an algorithmmodified from Automatch18, URANTEIN can search efficientlyfor uranyl-binding sites from these mutated pseudo-proteins.
Pible et al. designed an algorithm to identify native uranyl-binding proteins and the corresponding binding sites19. However,the Amber force-field-based scoring and optimizing algorithmused in their study was not suitable for searching the vastnumbers of mutations shown in our de novo design. The scoringfunction (see Supplementary Information) used in our programnot only predicts correct uranyl coordination geometry, but alsohelps to establish potential hydrogen-bonding interactions betweenuranyl oxo and residues from the binding protein. Structures ofuranyl complexes with inorganic or organic ligands8, as well ascrystal structures of proteins in which uranyl was used as a sourceof anomalous signal (Supplementary Table 3), provided a basis forthe screening and uranyl-binding protein designs.
Using URANTEIN we searched the PDB for pockets that couldaccommodate hexagonal bipyramid or pentagonal bipyramiduranyl-binding geometries, either natively or through mutation ofpotential ligand residues to aspartate/asparagine or glutamate/gluta-mine (see Supplementary Figs 2–4 and Supplementary Table 3). Weset the optimum distance between the oxygen-based ligand from theprotein to the central uranium ion at �2.46 Å. In addition, we alsoincorporated a search method for potential hydrogen bondingbetween the oxo groups of the uranyl with the scaffold protein inour screening process to maximize potential uranyl-binding affinityand selectivity (see Supplementary Information). From this initialscreen we identified over 5,000 hits. Hits were further selectedbased on their potential stability, potential steric clashes in the pre-dicted coordination site and accessibility of the binding site. Thecarboxylate side-chain ligand can be bidentate or monodentate,and therefore the fivefold or sixfold planar coordination aroundthe central uranyl can be flexible and affected by surroundingprotein residues. Ten promising candidates were selected, of
Solid support
Recovery
Adsorption
UO22+
UO22+
Zn2+
Ca2+
Cu2+
VO2+
Ni2+
Fe3+
Mg2+
Solid support
Figure 1 | Uranyl sequestration strategy. Immobilization of a high-affinity
and selective uranyl-binding protein on a solid support allows for the
enrichment of uranium over other metals. The protein was de novo designed
by computational screening based on the known scaffolds in the PDB.
Immobilized on resin or displayed on the cell surface by chemical or
biological means, the protein could effectively sequester uranyl from sea
water or uranyl-containing groundwater.
Pick a scaffoldCoordination features Build oxygen library and hydrogen library
Search for uranyl-binding sitesTop solutions library
Step 1 Step 2 Step 3
Step 4Step 5
Scoring and sorting
Go backto step 2if there is
no hit
Figure 2 | The main steps in computational screening and design of uranyl-binding proteins. In step 1, three uranyl-coordination features were designed. In
step 2, protein scaffolds were prepared by PDB query (version 2010). All entries that contained protein chains of length 60–200 amino acids (12,173 in total)
were employed as scaffolds. Then, one scaffold in the library was picked for further screening (for example, the green one shown here). In step 3, the oxygen
library of possible mutations and the hydrogen library of native protein were built (Supplementary Fig. 3). In step 4, for each scaffold, uranyl-binding sites
were searched using the oxygen library and hydrogen library. An algorithm called URANTEIN was used to complete this work efficiently (see Supplementary
Fig. 3 for the detailed algorithm). In step 5, the uranyl coordination geometries for the selected sites were evaluated and the results were filtered based on
the evaluating score. Finally, all the results were sorted based on the scores.
which nine proteins expressed well and four showed binding touranyl with Kd values of �100 nM and below (SupplementaryTables 4 and 5).
Development of super uranyl-binding protein (SUP). One ofthe hits stood out for its potential stability and utility, a smallprotein with unknown function from Methanobacteriumthermoautotrophicum, an anaerobe isolated from sewage sludge inUrbana, Illinois20. This protein consists of three a-helices in atight bundle (PDB accession 2PMR, Supplementary Fig. 4) and isthermally stable at room and elevated temperatures. In thecomputational model, Asp68, Asn17Glu, Leu13Asn and His64Glncoordinate with the uranium atom, and Arg71 forms a hydrogenbond with one uranyl oxo group. Three mutations of Leu13Asn,Asn17Glu and His64Gln were introduced in the wild-type protein(Supplementary Fig. 4). The mutant protein was cloned, expressedand purified. It exhibited a modest binding affinity for uranylwith a Kd of 37 nM (U09, see Supplementary Information). Basedon the model structure, we designed mutations that may increasethe binding affinity of U09. Although we were able to achieve amodest increase in binding affinity (Kd¼ 1.8 nM) by mutating anearby leucine to threonine (Leu67Thr) to stabilize the complexstructure, we were pleased to find that further mutations of thetwo neutral computational mutations His64Gln and Leu13Asn toGlu and Asp, respectively, led to an almost 106-fold increase ofthe uranyl-binding Kd to 7.4+2.0 fM at pH 8.9 (7.4 × 10215 M,Fig. 3a, Supplementary Table 5). At pH 6.0, the binding affinity ofSUP for uranyl decreases to Kd¼ 0.2+0.1 nM (SupplementaryFig. 5 and Supplementary Table 5), which implies that the tworesidues may contribute significantly to uranyl binding only athigher pH. The additional negative charge introduced by themutations could also help to stabilize binding of the positivelycharged uranyl.
As SUP exhibits a high binding affinity to uranyl, we examinedits selectivity for uranyl over that for other metal ions. Theprotein was immobilized on sulfhydryl resin (see SupplementaryInformation). Seventeen metal ions found in sea water and relevantto competition with uranyl were competed against uranyl forprotein binding (Fig. 3b, Supplementary Table 6). Most ions werenot able to outcompete uranyl, even at 2.0 × 106-fold excess. For
instance, SUP showed selectivity for uranyl greater than or equalto 2 × 106-fold over calcium(II). To our knowledge such a highselectivity is unprecedented for proteins. For those ions that didcompete, none competed at concentrations low enough to interferewith binding to uranyl in sea water. In fact, the only metals that wecould show to compete are Cu2þ at 103-fold excess and vanadyl(VO2þ) at 104-fold excess. The concentration of Cu2þ is roughly2.4 nM in sea water and the concentration of vanadium is about40 nM, and therefore neither could significantly affect the uranylbinding. The high affinity for Cu2þ may not be surprising giventhe overall stability of Cu2þ complexes as described in the Irving–William series. The vanadyl ion has similarities in structure andcharge to the uranyl ion, with only a slightly smaller covalentradius, which suggests a binding motif similar to that of uranyl.
Crystal structure of SUP. To investigate further the mechanism ofuranyl binding by SUP, we crystallized both uranyl-bound SUP andthe apo-SUP at pH 4.0 (Fig. 4, Supplementary Table 7). The high-resolution crystal structure of the uranyl–protein complex showeda pentagonal bipyramidal binding configuration similar to theinitial computational model (Fig. 4b,d). The uranium positiononly deviates by 0.47Å (Fig. 4d). Three of the designed ligandresidues (Glu17, Asp68 and Arg71) interact with uranyl to showside-chain conformations similar to the predicted conformations.Major deviations occur in the modifications of Asn13Asp andGln64Glu designed post-screening, as these two residues are notdirectly involved in uranyl binding in the solved structureobtained under acidic conditions. We cannot exclude thepossibility that these residues may bind to the central uranylunder the basic conditions used for binding assays and uranylsequestration from sea water; however, we have yet to crystallizethe protein under basic or neutral conditions. Overall, the crystalstructure of the uranyl–protein complex supports the validity ofthe URANTEIN algorithm as an effective tool for the initialscreen. Binding of uranyl at the interface of three monomers wasalso observed (Supplementary Fig. 6). Most probably, this site isformed during the crystallization process because the proteinforms a stable dimer in solution, whether the uranyl ion ispresent or not (Supplementary Fig. 7). In addition, this secondarybinding site does not appear to be important in the binding of
Figure 3 | Uranyl-binding affinity and selectivity of SUP. a, Competition assay of SUP versus total carbonate for uranyl yields a Kd of 7.4 fM at pH 8.9. The
final solution of each point contained 10 mM protein, 10mM UO22þ and different concentrations of carbonate (fit parameters: maximum¼0.992+0.013,
minimum¼0.033+0.010, midpoint¼0.398+0.018 mM, Hill coefficient¼ 1.72+0.11, x2red¼0.457, R2¼0.9934). b, Binding selectivity of SUP for uranyl
over various other metals relevant to sea water extraction. Hatched columns, molar excess of ions in sea water; filled columns, selectivity of metal ions to
uranyl by competition assay. The maximum molar excess tested was 4 × 106 for most metals because of the solubility of the salts and limits of detection of
the assay. For mercury and lead, the maximum molar excess tested was 105 (for experimental details, see Supplementary Information and Supplementary
Table 6). The standard deviations were calculated from the uncertainty in the measurement of uranyl concentration.
uranyl, as titration of 10 mM SUP with uranyl shows the presence ofa single strong binding site (Supplementary Fig. 5).
Structural features explain the high uranyl-binding affinity andselectivity of SUP, which contains four mutations (Asp13, Glu17,Glu64 and Thr67), compared to the original template protein:
† Residues Glu17 and Asp68 directly bind the uranyl ion in theequatorial plane. Mutations of Glu17Gln or Asp68Asn dramati-cally decreased the binding affinity, which supports these resi-dues as being ligands to uranyl. The remaining equatorialbinding site is occupied by a water molecule.
† Asp13 and Glu64 provide a negatively charged environment tohold the uranyl ion (Fig. 4c). The negative charge is a seeminglyessential attribute because the neutral mutant homologues(Asn13, Gln64) show much lower affinity. These residues couldbind uranyl directly under basic conditions.
† Arg71 forms a hydrogen bond with an axial oxo of the uranylion in the complex structure, but in the apo form it forms a saltbridge with Glu17 (Fig. 4b, Supplementary Fig. 8). This inter-action contributes to the high selectivity for the uranyl ionthrough recognition of the oxo group. Vanadyl(IV), likeuranyl, is an oxo cation with a þ2 charge and can competewith uranyl at the 104-fold level, which suggests that Arg71may also form a hydrogen bond with the single oxo groupof vanadyl.
† Leu67Thr stabilizes the binding state via hydrogen bonding withbackbone Ala63. Thr67 also better accommodates the bending ofthe last helix on uranyl binding compared to that of leucinebecause threonine is a residue known to destabilize the helixstructure (Supplementary Fig. 9).
Uranyl sequestration from sea water. To confirm the function ofthis uranyl-binding protein, we tested uranyl-sequestrationefficiency. More than 95% of the uranyl was removed aftertreating uranyl-containing water (666 nM uranyl, 158 parts per109 (ppb) at pH 7.0) with SUP fused with a maltose-bindingprotein (MBP-SUP) immobilized on amylose resin (seeSupplementary Information). The residual uranyl concentrationwas 3–8 ppb, far lower than the 30 ppb US EnvironmentalProtection Agency standard for drinking water in the USA. Theresin can be recycled easily by washing with carbonate solutionand the efficiency was retained after many cycles (Fig. 5a).
Next, we applied MBP-SUP to test uranyl sequestration fromsynthetic sea water. Synthetic sea water was prepared by amethod, reported previously, to account for all major ions andanions above 1 mM, with uranyl present at 13 nM (ref. 21). The
fusion protein bound to amylose resin showed a good efficiency,with �17% of the uranyl sequestered with one equivalent ofimmobilized protein used against one equivalent of uranyl(13 nM) in the corresponding amount of sea water in a very shortincubation time of 30 minutes (Fig. 5b), whereas ten equivalentsof the immobilized protein sequestered �30% of the uranyl fromthe same amount of synthetic sea water. When an excess of SUPproteins (.6,000 equiv.) was used we were able to remove over90% of the uranyl (Supplementary Fig. 10), similar to recentresults with a layered solid-state ion-exchanger approach12, andonly less than 5% of the uranyl remained bound to protein-freeamylose resin. We also used the Escherichia coli surface display asa complimentary example of sea water sequestration. SUP proteinwas displayed on the E. coli surface using an established OmpAfusion method17. The protein was highly expressed and displayedon the cell surface (see Supplementary Fig. 11). This system iscapable of extracting more than 60% of the uranyl in synthetic seawater (Fig. 5b). The engineered protein is stable thermally with aTm of �71 8C (see Supplementary Fig. 12) and the bacterialsystem can be regenerated easily, which thus offers an economicsource for uranyl sequestration. To our knowledge, this is the firstbiological system that can effectively sequester uranyl from sea water.
DiscussionOwing to active human activities, natural metal resources have beenexploited at an accelerated pace with increasing metal pollution.Thus, new technologies that can sequester and remediate metals effi-ciently from dilute environmental sources and sites are highly desir-able. Uranyl, the predominant aerobic form of uranium, is presentin the ocean at a concentration of 13.7 nM. To sequester uranyleffectively from sea water requires the development of uranyl-binding ligands with femtomolar or higher affinity for uranyl andexceedingly high selectivity over calcium(II) (.1 × 106-fold) andother abundant metal ions present in the ocean. Currently, no exist-ing small-molecule or protein-based ligands can achieve thisdaunting task.
We present here a strategy to design and develop selective metal-binding proteins starting from a computational screening for suit-able scaffolds. Applications of de novo protein design22–24 as wellas rational design based on existing protein scaffolds have led tometalloproteins with new functions25–31. A rapidly expandingknowledge of protein structure and function has allowed researchersto employ effective strategies that can incorporate active sitesranging from catalytic sites to binding hot spots and metal-coordi-nation patterns27 into new scaffolds for designing novelenzymes32,33, target protein binders34,35 and metalloproteins36,37.
Arg71
Asp68Glu172.9
2.9
2.7 2.4
3.12.3
Crystal structure
Design model
Lys9Asp13
Asn13
Arg71
Asp68
Glu17
Glu64
Gln64 Thr67
w124
Helix I
Helix II
Helix III
a b c d
Figure 4 | Uranyl–SUP crystal structure. The high-resolution crystal structure of the uranyl–protein complex reveals the metal binding site that is close to the
computational prediction. The uranyl ion is in a negatively charged pocket with a pentagonal bipyramidal binding configuration. a, Overview of the crystal
structure of the SUP–uranyl complex. b, Uranyl-binding pocket detail of SUP. c, SUP surface electrostatics (blue colour for the positive regions and red colour
for the negative regions). d, Comparison of SUP design model (magenta) to SUP crystal structure (blue).
Central to this strategy are the determination of active-site structuresand the scaffold selection. Although the incorporation of metal-binding sites to both de novo and native scaffolds has beenstudied comprehensively36, nearly all of these metalloprotein-design cases utilized either known scaffolds from existing or denovo designed metalloprotein templates. Although large-scale scaf-fold selection has been employed successfully in enzyme andprotein–protein interaction designs32–37, such a selection strategyhas yet to be applied for metalloprotein designs.
We have shown that a de novo design program can be developedto search for specific sites in scaffold proteins that may accommo-date the pentagonal or hexagonal bipyramidal geometry thatbinds uranyl selectively from most other metal ions. The subsequentrational design based on structural and binding experiments(Supplementary Fig. 2) led to a stable protein that exhibits femto-molar affinity and unprecedented selectivity towards uranylbinding. This robust system, which can be regenerated on bacterialsurfaces or reused on the resin, is capable of practically enrichinguranyl from sea water and both avoids competition from majorions and repeatedly cleansing uranyl-containing water. Althoughother approaches to extract uranyl from sea water have been pro-posed, and even implemented, our protein-based system mayaddress three limitations present in current strategies: (1) cost, (2)selectivity and (3) adaptability:
(1) The genetically encoded system described here may be biologi-cally regenerated and displayed on the surface of living bacteriaor in plants, with minimal costs. The bacteria or plants may beused directly for uranyl enrichment.
(2) The superb selectivity is characteristic of protein-based binding.Such a high selectivity would be cost prohibitive to achieve withlarge-scale synthetic systems based on chelator or ion exchange.Once developed, the superb selectivity and the high binding affi-nity of the current system are genetically encoded and readilyregenerated in living host systems. Other approaches may notmatch the selectivity of our protein-based system in the presenceof many other metal ions in sea water.
(3) Furthermore, with a genetically encoded system, directed evol-ution could be employed in the future to improve the perform-ance further and to make the system adaptable to various
conditions required for potential applications. The constructswe describe open up broad possibilities for the extraction ofuranyl and for biological remediation. The strategy presentedhere may be applied to engineer protein-based reagents forselective metal binding in metal sequestration and remediation.
MethodsComputational design of uranyl-binding proteins. All entries from the PDB thatcontain protein chains of length 60–200 amino acids (12,173 in total) wereemployed as scaffolds. Uranyl coordination was generated based on uranyl-containing protein structures from the PDB and uranyl-binding molecules from theCambridge Crystallographic Data Centre (CCDC). The oxygen ligands in theequatorial plane of the hexagonal bipyramidal or pentagonal bipyramidal uranylcould come from main-chain amides, water, natively or mutationally generatedaspartate/asparagine or glutamate/glutamine; mutations were generated using thebasic Mayer rotamer library. A program named URANTEIN was developed tosearch the protein scaffold library efficiently for pockets that might accommodateuranyl. The program obtained all possible coordinating oxygens (which generatedan oxygen library), and then searched for uranyl-binding sites using the library (seedetails in Supplementary Information and Supplementary Fig. 3). The uranyl-binding sites were evaluated by a scoring function that contained oxygencoordination, oxygen compatibility and hydrogen bonding. The best-scoringsolutions were checked further manually with consideration of steric clash, the depthof uranyl in its binding pocket and the stability of the scaffold protein.
Protein purification and crystallization. The gene sequences of U01-U10 and SUP-OmpA fusion protein were synthesized by GeneScript. Mutations were performedusing Pfu Ultra II polymerase from Agilent. U09 and mutations were cloned andexpressed in pMCSG19 vector for expression in BL21 (DE3) pRK1037 E. coli strains.The strains that carried the plasmids were grown in LB (Lysogeny Broth) toattenuance (D600)¼ 0.6, induced with 1 mM IPTG, and cells were grown overnightat room temperature before harvesting. Cells were lysed by sonication in the lysisbuffer that contained 20 mM Tris-HCl (pH 7.4), 500 mM NaCl and 1 mMdithiothreitol (DTT) in the presence of 1 mM phenylmethyl sulfonyl fluoride as theserine protease inhibitor. Supernatant was separated by centrifugation and loaded onnickel nitrilotriacetic acid columns. Protein was obtained using 10 mM Tris pH 7.4,500 mM NaCl with 1 mM DTT as elution buffer and imidazole ramping from 0 to500 mM. The His6-tag was removed by tobacco etch virus (TEV) protease. Allcrystallization samples were further purified by gel filtration in buffer containing10 mM Tris pH 7.4, 100 mM NaCl and 1 mM DTT. Crystals were produced usinghanging-drop vapour diffusion at 16 8C by mixing 1 ml protein solution at 10–20 mgml21 with a 1 ml reservoir solution that contained 2% v/v Tacsimate pH 4.0, 0.1 Msodium acetate trihydrate pH 4.6, 16% polyethylene glycol 3350. For the SUP–uranylcomplex, uranyl was mixed with SUP at a 1.2:1 molar ratio before the drop set.
Uranyl-binding assay. A modification of the Arsenazo III method was employed todetermine uranyl concentrations. Arsenazo III) (80 mM, 50 ml) that contained 0.1 M
0
20
40
60
80
100
Per
cen
t sea
wat
er r
ecov
ered
(%
)
Fusion proteinto uranyl 1:1
Fusion proteinto uranyl 10:1
Cell surfacedisplay
1 2 3 4 5 6 7 8 90
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100a b
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Figure 5 | Immobilized SUP provides a useful manifold for a variety of applications. a, SUP immobilized on amylose resin can consistently remove uranyl
from uranyl-contaminated water over many cycles. These experiments were performed in pH 7.0 buffer to mimic groundwater. The standard deviations were
calculated from the uncertainty in the measurement of uranyl concentration. b, Both protein display by fusion with maltose-binding protein and
immobilization on amylose resin and by fusion with OmpA and display on live E. coli cells allow for effective recovery of uranyl in synthetic sea water21. The
use of one and ten equivalents of resin-immobilized protein against one equivalent of uranyl (13 nM) in the synthetic sea water can sequester �17% and
�30% of the total uranyl, respectively. Over 60% of the uranyl can be sequestered with the use of surface-displayed E. coli cells. The standard deviations
HCl was titrated with an equal volume of uranyl solutions ranging from 0 to 30 mM,and the absorbances at 652 nm and 800 nm were monitored. The values of A652 andA800 increased linearly in this range, and can be converted into uranylconcentrations. Diglycolic acid (DGA) and carbonate competition assays were usedto determine the Kd values of uranyl-binding protein. DGA competition assays wereperformed at pH 6.0 or 6.5 in 10 mM Bis-Tris buffer with 300 mM NaCl. Standardsolutions of 100 mM protein and 100 mM UO2
2þ were prepared and diluted tenfoldwith the appropriately scaled DGA buffer. The final solutions contained 10 mM Bis-Tris (pH 6.5), 10 mM protein, 10 mM UO2
2þ and different concentrations of DGA.The solutions were mixed and filtered through centrifuge filters with a 3 kDa cutoff.Flow through was tested for uranyl concentration using an Arsenazo III assay.Carbonate competition assays were performed similarly to the DGA assays, but withfreshly prepared pH 8.0–9.0 carbonate solutions in Tris-HCl buffer. All the waterused to prepare solutions was freshly degassed, deionized and protected againstfurther sorption of atmospheric CO2. The Kd of the uranyl–protein complex wasdetermined by fitting the resulting binding curve using the uranyl–DGA or uranyl–carbonate binding constants.
Sea water sequestration assays by amylose resin or surface-display cells. For thepreparation of SUP-bound amylose resin, SUP was cloned into pMCSG19 vectorand expressed in BL21 E. coli to produce a MBP fusion protein. The protein was thenincubated with amylose resin to obtain SUP-bound resin. For the preparation ofsurface-displayed cells, the SUP-OmpA fusion protein was cloned into pBAD vectorand expressed in BL21 E. coli, with a linker GGGSGGGS between SUP and OmpA.Cells were grown to D¼ 0.6 in LB medium and induced by 2% L-arabinoseovernight, and harvested by centrifugation at 5,500g. For the sea water extractionassay, SUP-bound amylose resin (5 ml) or surface-display cells (0.5 ml pellet) wereincubated with 50 ml synthesized sea water for 30 minutes at room temperature.Supernatants were sent for uranium analysis by inductively coupled plasmamass spectrometry.
Received 7 November 2013; accepted 17 December 2013;published online 26 January 2014
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AcknowledgementsThis work was supported by the Division of Chemical Sciences, Geosciences, andBiosciences, Office of Basic Energy Sciences of the US Department of Energy, undercontract number DE-FG02-07ER15865 to C.H., and at Argonne National Laboratory(M.J.) under contract number DE-AC02-06CH11357, the Dreyfus FoundationPostdoctoral Program in Environmental Chemistry to S.O., the Ministry of Science andTechnology of China (2009CB918500) and the National Natural Science Foundation ofChina (21173013, 11021463) to L.L. Use of the Advanced Photon Source for proteincrystallography data collection at beamlines LS/CA-CAT (21-ID-F) and NE-CAT(24-ID-C) was supported by the Office of Basic Energy Sciences of the US Department ofEnergy under contract number DE-AC02-06CH11357. We thank S. F. Reichard forediting the manuscript and C. Yang and L. Lan for experimental support.
Author contributionsC.H. conceived the project. C.H. and L.L. designed the experiment. C.Z. and L.L. performedthe initial screening. W.Z. and C.J.L. performed subcloning of virtual hits. S.O. and C.J.L.expressed, purified and tested all first-generation virtual hits. L.Z., S.O. and M.B. designedsecond-generation mutants. L.Z., M.B. and J.L. expressed and tested all protein derivativesand designed all later-generation mutants. L.Z., M.B., M.P.J. and C.H. analysed the data andM.B. and L.Z. co-wrote the manuscript. All authors discussed the results and commentedon the manuscript.
Additional informationSupplementary information is available in the online version of the paper. Reprints andpermissions information is available online at www.nature.com/reprints. Correspondence andrequests for materials should be addressed to L.L. and C.H.
Competing financial interestsA patent application has been filed for the technology disclosed in this publication.
