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A selective, protein-based fluorescent sensor with picomolar affinity for rare earth elements Joseph A. Mattocks, Jackson V. Ho, and Joseph A. Cotruvo, Jr. J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 6, 2019
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Journal of the American Chemical Society
A selective, protein-based fluorescent sensor with picomolar affinity for rare earth elements Joseph A. Mattocks, Jackson V. Ho, and Joseph A. Cotruvo, Jr.* Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States
Supporting Information Placeholder ABSTRACT: Sensitive yet rapid methods for detection of rare earth elements (REEs), including lanthanides (Lns), would facilitate mining and recycling of these elements. Here we report a highly selective, genetically encoded fluorescent sensor for Lns, LaMP1, based on the recently characterized protein, lanmodulin. LaMP1 displays a 7-fold ratiometric response to all LnIIIs, with apparent Kds of 10-50 pM, but only weak response to other common divalent and trivalent metal ions. We use LaMP1 to demonstrate for the first time that a Ln-utilizing bacterium, Methylobacterium extorquens, selectively transports early Lns (LaIII–NdIII) into its cytosol, a surprising observation as the only Ln-proteins identified to date are periplasmic. Finally, we apply LaMP1 to suggest the existence of a LnIII uptake system utilizing a secreted metal chelator, akin to siderophore-mediated FeIII acquisition. LaMP1 not only sheds light on Ln biology but also may be a useful technology for detecting and quantifying REEs in environmental and industrial samples.
Rare earth elements (REEs), including the lanthanides (Lns), are important components of many current technologies, from permanent magnets and lasers to smartphones and electric cars, with a global demand of 149,000 metric tons in 2015.1-2 However, access to these elements to meet this demand is limited by insolubility of LnIII salts in the presence of common ions such as phosphate (10-13 M),3 necessitating expensive, low-throughput detection methods and difficult, environmentally unsustainable isolation and separation procedures.4 These challenges have motivated research into new strategies to detect5-6 and extract and separate7-9 Lns from feedstocks. These approaches have been bolstered by the recent discovery that many methylotrophic bacteria, such as Methylobacterium extorquens, specifically incorporate Lns into one of their metabolically most important enzymes, pyrroloquinoline quinone-dependent methanol dehydrogenase (MDH).10-13 Many questions remain regarding Ln utilization by these organisms: how these bacteria acquire Lns, why (and how) only early Lns, primarily La-Nd, support Lndependent growth,6,11,14-16 whether Lns are limited to the periplasm where MDH resides or can also access the cytosol, and how many Ln-dependent proteins exist. Answers to these questions would aid in development of more sustainable bioengineering methods for Ln detection and concentration, for example enabling the utilization of feedstocks with lower than the ~300 ppm (2 mM) minimum total REE standard currently used.2 We recently reported discovery17 and biochemical and structural18 characterization of lanmodulin (LanM), a 12-kDa
protein that uses coordination motifs traditionally associated with CaII binding (EF-hands) for high-affinity LnIII binding. This protein, found in the periplasm of M. extorquens, binds 3 equiv. LnIIIs and YIII with apparent Kds of 5-25 pM, and 1 equiv. with approximately micromolar affinity; a large conformational change occurs upon metal ion binding with 108-fold selectivity for LnIIIs over CaII. These unique properties inspired us to utilize LanM as the basis for a ratiometric protein-based (genetically encoded) fluorescent sensor19-24 for Lns (Figure 1). Here we present this sensor, LaMP1 (lanmodulin-based protein sensor 1), the first of its type of Lns, and use it to provide new insights into Ln uptake in M. extorquens. Our work suggests that this and related constructs may be broadly useful biotechnologies for REE detection in environmental and biological samples.
Figure 1. Design of LaMP1. Fluorescent protein images are derived from PDB entry 1CV7 using PyMOL. In order to construct LaMP1, we started with the calmodulinbased sensor D2, developed by Tsien and coworkers,19 which includes a C-terminally truncated enhanced cyan fluorescent protein (ECFP) and citrine, a yellow fluorescent protein, as a Förster resonance energy transfer (FRET) pair. We replaced the calmodulin and M13 peptide components of D2 with LanM(Ala22-Arg133),17 yielding LaMP1 (Figure 1, Figure S1). LaMP1 exhibits a strong, 7-fold ratiometric FRET response to all REEs (Figure 2a,2d), with apparent dissociation constants (Kd,app) for LnIIIs and YIII in the picomolar range (Figure 2c, Figure S2, and Table S1), consistently only ~2-fold weaker than those of native LanM.17 The Kd,app of LaIII-LaMP1 measured by circular dichroism spectroscopy (Figure S3) agrees with the FRETderived value, suggesting that LaMP1 undergoes a similar conformational change as LanM itself. Our method for titrating the chelator used for these determinations, ethylenediamine N,Nꞌdisuccinic acid (EDDS), could not be used to calibrate solutions of Tm, Yb, and Lu (see SI Methods); however, we anticipate Kd,app values of ~40-50 pM based on the relationship between Kd,app and ionic radius in Figure 2c. In the case of LaIII, we tested fluorescence response over the full range of free metal concentrations from 10-13 M to 10-2 M, buffered using EDDS or trimethylenedinitrilotetraacetic acid (TMDTA), or without
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chelator (Figure 2b). No significant additional fluorescence response was observed above 10-10 M until the unbuffered regime (>1 µM), which we attribute to metal binding to EF-hand 4 based on our previous analyses of LanM.17-18
Figure 2. LaMP1 is a robust, selective fluorescent sensor for LnIIIs. (A) Response of 500 nM LaMP1 to 0–4 (black – red) equiv. LaIII (λex = 433 nm). (B) FRET ratio (F529nm/F475nm) of LaMP1 as a function of free LaIII concentrations either EDDS- or TMDTA-buffered or unbuffered. (C) Plot of apparent Kd of LnIII-LaMP1 versus ionic radius25 for LaIII–ErIII and YIII. (D) Metal selectivity of LaMP1. Fold response of 500 nM LaMP1 to 5 µM LaIII–LuIII; 10 µM ScIII, YIII, and FeIII; 100 µM CuII; or 1 mM AlIII, MnII, and CaII. LaMP1 also exhibited fluorescence response to CaII, but with a Kd,app of 1.2 mM, far above likely physiological levels, and a FRET response of just 3-fold (Figure S4, Table S1). We note, however, that this determination was limited by fluorescence quenching at CaII concentrations >10 mM, which may have obscured a further FRET increase. We also tested LaMP1 against common metal ions – FeIII, AlIII, MnII, and CuII – at concentrations that are potentially relevant to aqueous environmental samples such as mine leachates,9 and found little or no response (Figure 2d). At higher concentrations, FeIII quenched fluorescence and the sensor showed modest response to AlIII and MnII, but all were outcompeted by 5 µM LaIII (Figure S5). Biologically relevant ions MgII, Na+, and K+ also minimally interfere (Figure S5). Therefore, like LanM itself, LaMP1 exhibits high selectivity for LnIII over CaII and other common metal ions. LaMP1’s limit of detection in plate reader fluorescence assays (5 nM LaMP1) was 10 nM LaIII, within 1 min (Figure S6). LaMP1’s performance compares favorably with previously reported approaches to selectively detect Lns. He and co-workers5 engineered a bacterial two-component system to detect Lns via incorporation of a lanthanide binding tag26 peptide sequence. After 6 h of growth, this system exhibits 3-fold fluorescence response to 1 µM TbIII and a smaller but significant response to
200 nM TbIII, but also responds 2-fold to 50 µM CaII. Skovran, Martinez-Gomez, and co-workers have recently reported an M. extorquens strain with a fluorescent protein gene under control of the xox1 promoter, which responds to 2.5 nM LaIII but requires several hours of incubation; furthermore, this promoter only responds to La–Sm.6 A colorimetric method based on the arsenazo-III dye has a detection limit of ~500 nM but only responds reliably to La–Eu.27 Therefore, LaMP1 is a uniquely rapid, selective, and highly sensitive tool for detecting all REEs. LaMP1’s robust performance in vitro suggested that it might enable us to probe LnIII levels and localization within M. extorquens. In particular, we aimed to address two important outstanding questions: 1) whether Lns are limited to the periplasm, where the only presently identified lanthanoproteins (XoxF,10 ExaF,28 and LanM17) are localized, and 2) why only the lighter Lns, La–Sm, are able to support Ln-dependent growth of M. extorquens on methanol. These are challenging questions to address using conventional methods as M. extorquens is not amenable to fractionation of periplasm and cytosol.29 As the presence of periplasmic Lns has been established by characterization of several Ln-dependent alcohol dehydrogenases10-11,13,28,30-32 and of LanM,17-18 we probed cytosolic Ln uptake by expression of LaMP1, under control of the strong mxaF promoter,33 in M. extorquens. Cells were grown with
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Journal of the American Chemical Society CaII in the absence of Lns, yielding a cellular FRET ratio similar to that of apo-LaMP1 in vitro. Upon addition of 2 µM LnIII to the medium, LaMP1 fluorescence was monitored over 10 h (~2 doublings) of further growth. To our surprise, the FRET ratio of LaMP1 was increased selectively and significantly by La, Ce, Pr, and Nd within 2 h (Figure 3, Figure S7), suggesting cytosolic uptake, but heavier Lns did not yield a significant response versus the Ca control, except for Sm at ≥7 h. Importantly, as LaMP1’s Kd,app values for LnIIIs La–Tb are not significantly different, the negligible in-cell response of the sensor to later Lns strongly suggests that Lns beyond Nd are not efficiently transported to the cytosol.
Figure 3. Kinetics of cytosolic LnIII uptake by M. extorquens, monitored using LaMP1. M. extorquens cells expressing LaMP1 were grown at 30 °C without Lns (with 20 µM Ca). At t=0, 2 µM each LnIII was added to individual cultures and FRET ratio was monitored over 10 h. Data points are mean ± SEM for 5 independent experiments. *p
