Uranium is a key element used in nuclear energy production and is crucial in many other applications. The most stable and relevant uranium ion in aerobic environments is the uranyl cation. Super Uranyl-binding Protein(SUP) has been rationally designed via structural calculations and functional modification to specifically bind uranyl cations. According to the researchers’ results, SUP is thermodynamically stable and offers very high affinity and selectivity for uranyl with a Kd of 7.4 fM and >10,000-fold selectivity over other metal ions1. The binding features of SUP are described later in more detail (Fig.1).
It was found that UO22+ is coordinated by five carboxylate oxygen atoms from four amino acid residues in SUP. The hydrogen bonds between the amino acid residues coordinating UO22+ and residues in its second coordination sphere also affects the protein’s uranyl binding ability (Fig.2) 2.
As mentioned above, we fused SUP to three SpyTags in order to construct the 3A-SUP monomer, so the function of SUP might be affected by these additional modules. To make sure that 3A-SUP could still function when bound in the hydrogel, we tested its ability to adsorb UO22+ in various environments.
Appropriate volumes of 3/4/6A-SUP and 3B were mixed and incubated for 1 hour. Subsequently, a uranyl solution in TBS buffer was prepared and its pH value adjusted appropriately. After complete cross-linking, the uranyl solution was contacted with the pre-incubated proteins and mixed thoroughly by vortexing (Fig.3).
The adsorption reaction was allowed to continue for 1 min, after which the mixture was immediately transferred into 10kDa cutoff centrifuge filters and centrifuged for 10 min at 14000g to exclude non-specific protein interference by removing proteins (Fig.4). Finally, 100μL aliquots of the filtrate were collected for further analysis.
What makes these results more reliable is that we set up control groups which contained the same concentration of uranyl as the test groups. We used the uranyl concentration of the filtrates from the control groups as the actual uranyl concentration to account for the adsorption of uranyl on centrifuge filters. Two different methods were applied to determine the uranyl concentrations in the filtrate. For higher concentrations (>1μM), we used a modification of the Arsenazo III method1. For lower concentrations (<1μM), ICP-MS was employed.
The adsorption capacities of 10μM proteins and hydrogel were measured in TBS buffer against uranyl in equimolar quantities. In very short time, 3A-SUP alone showed an adsorption capacity of up to 87% and cross-linked 3A-SUP+3B could effectively sequester 96% of the total uranyl (Fig. 6).
Furthermore, we measured the adsorption capacities of other kinds of fusion constructs and polymers, such as 4A-SUP (84%), 6A-SUP (87%), 4A-SUP+3B (88%), 6A-SUP+3B (63%) (Fig. 7). All of these were less efficient but they also could sequester at least 60% of the total uranyl. The standard deviations were calculated from triplicate experiments (n=3). These results were promising since different kinds of fusion proteins and protein constructs were able to adsorb uranyl with great efficiency. Among these, 3A-SUP+3B (96%) showed the best adsorption capacity and was consequently used for all further experiments.
We next wondered whether the adsorption capacity of 3A-SUP+3B would increase with an increased protein-uranyl ratio. We thus tested the adsorption capacity of 3A-SUP+3B at different protein-uranyl ratios in TBS buffer. In these experiments, the concentration of uranyl ions was kept at 10μM.
The 3A-SUP+3B hydrogel could sequester 89% and 93% of the total uranyl when the protein to uranyl ratio was one and ten, respectively (Fig.8). The standard deviations were calculated from triplicate experiments
The results thus showed that when the protein-uranyl ratio was increased tenfold, the adsorption rate increased as well. However, the rate of increase was less than 5% and was furthermore not statistically significant.
According to these results, the adsorption rate did not increase significantly when the protein to uranyl ratio was increased to ten. The reason might be that 3A-SUP+3B could sequester almost all of the available uranyl already when the ratio is 1:1, making the increase inconsequential. In conclusion, a protein-uranyl ratio of 1:1 was enough to sequester most uranyl at a concentration of 10μM.
The protein constructs showed a great uranyl adsorption capacity in TBS buffer, therefore we wondered whether they could work just as well in other conditions. We prepared two experimental scenarios simulated real-life pollution, using fresh water from Weiming Lake on campus and artificial seawater, respectively.
At the protein-uranyl ratio of 10:1, 3A-SUP+3B could sequester 93%, 67% and 83% of total uranyl in TBS buffer, fresh water and artificial seawater, respectively (Fig.9). The standard deviations were calculated from triplicate experiments.
When 3A-SUP+3B was used in fresh water, the adsorption capacity dropped sharply. It is possible that carbonate and other species found in the fresh water might have interfered with 3A-SUP+3B and led to the decrease. It is also possible that the proteins are unstable under low-salt conditions, as opposed to the NaCl-rich environments of TBS buffer or artificial seawater.
The adsorption capacity of 3A-SUP+3B against 13nM uranyl was tested at a protein-uranyl ratio of 6000:1, and it was able to sequester 48% and 35% of the total uranyl in TBS buffer and artificial seawater, respectively (Fig. 10). That means that the functional hydrogel could not only adsorb uranyl at high concentrations of uranium pollution, but could also bind uranyl at very low concentrations - as low as 13nM - which equals the uranyl concentration in natural seawater.
Triple SpyTag-SUP (3A-SUP) was able to adsorb uranyl with great efficiency, and the cross-linked product of 3A-SUP+3B showed an even better adsorption capacity. What’s more, 3A-SUP+3B could also perform its function well in contaminated conditions, and could thus be used to detoxify the environment. Furthermore, we confirmed that the biological functional hydrogel could indeed adsorb uranyl ions from seawater, and might even be employed to gather uranium directly from seawater in the future. Thus, our biological functional hydrogel has broad potential application fields.
References:
[1] Lu, Z. et al. A protein engineered to bind uranyl selectively and with femtomolar affinity. Nature chemistry 1856, 236-241 (2014).
[2] Laura, G, et al. UO22+ Uptake by Proteins: Understanding the Binding Features of the Super Uranyl Binding Protein and Design of a Protein with Higher Affinity. J. Am. Chem. Soc 136, 17484−17494 (2014).
