Anal. Chem. 2008, 80, 5750–5754
Spectroscopic Analysis of Ligand Binding to Lanthanide-Macrocycle Platforms James P. Kirby,*,† Morgan L. Cable,†,‡ Dana J. Levine,†,‡ Harry B. Gray,‡ and Adrian Ponce*,†,‡ Beckman Institute, California Institute of Technology, Pasadena, California 91125, and Planetary Science Section, Jet Propulsion Laboratory, Pasadena, California 91109 A high-affinity, binary Eu3+ receptor site consisting of 1,4,7,10-tetraazacyclododecane-1,7-diacetate (DO2A) was constructed with the goal of improving the detection of dipicolinic acid (DPA), a major component of bacterial spores. Ternary Eu(DO2A)(DPA)- complex solutions (1.0 µM crystallographically characterized TBA · Eu(DO2A)(DPA)) were titrated with EuCl3 (1.0 nM-1.0 mM); increased Eu3+ concentration resulted in a shift in equilibrium population from Eu(DO2A)(DPA)- to Eu(DO2A)+ and Eu(DPA)+, which was monitored via the ligand field sensitive 5D0 f 7F3 transition (λem ) 670-700 nm) using luminescence spectroscopy. A best fit of luminescence intensity titration data to a two-state thermodynamic model yielded the competition equilibrium constant (Kc), which in conjunction with independent measurement of the Eu(DPA)+ formation constant (Ka) allowed calculation of the ternary complex formation constant (Ka′). With this binding affinity by competition (BAC) assay, we determined that Ka′ ) 108.21 M-1, which is ∼1 order of magnitude greater than the formation of Eu(DPA)+. In general, the BAC assay can be employed to determine ligand binding constants of systems where the lanthanide platform (usually a binary complex) is stable and the ligand bound versus unbound states can be spectroscopically distinguished. The anthrax attacks of 2001 prompted us to develop new methods for the rapid detection of bacterial spores.1–3 Our strategy is based on the fact that a large increase in airborne bacterial spore concentration is a strong indication of an anthrax attack, as Bacillus anthracis weaponized spore powders were employed. In addition, bacterial spores are extensively employed as biological indicators for monitoring the efficacy of sterilization processes.4 * To whom correspondence should be addressed. E-mail: james.p.kirby@ jpl.nasa.gov;
[email protected]. † Jet Propulsion Laboratory. ‡ California Institute of Technology. (1) Sanderson, W. T.; Stoddard, R. R.; Echt, A. S.; Piacitelli, C. A.; Kim, D.; Horan, J.; Davies, M. M.; McCleery, R. E.; Muller, P.; Schnorr, T. M.; Ward, E. M.; Hales, T. R. J. Appl. Microbiol. 2004, 96 (5), 1048–1056. (2) Jernigan, J. A.; et al. Emerg. Infect. Dis. 2001, 7 (6), 933–944. (3) Yung, P. T.; Lester, E. D.; Bearman, G.; Ponce, A. Biotechnol. Bioeng. 2007, 84 (4), 864–871. (4) Albert, H.; Davies, D. J. G.; Woodson, L. P.; Soper, C. J. J. Appl. Microbiol. 1998, 85 (5), 865–874.
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Since each bacterial spore contains more than 108 molecules of dipicolinic acid (DPA),5 rapid detection is enabled via DPAsensitized lanthanide luminescence.6–12 The electronic excited states of Eu3+ and Tb3+ ions possess long lifetimes (0.1 to over 1 ms); the corresponding absorption bands have small extinction coefficients (ε ∼ 1 M-1 cm-1); and the emission features are narrow.13 Binding of an organic chromophore such as DPA (ε ∼ 5000 M-1cm-1 8) triggers intense luminescence under UV excitation. This luminescence turn-on is facilitated by an absorption-energy transfer-emission process,14–18 which results in a large apparent Stokes shift. Due to these properties, DPA-sensitized lanthanide luminescence is very sensitive and applicable to time-gated detection, which essentially eliminates the backgrounds from environmental samples that often render fluorescence methods useless. In an effort to increase the sensitivity of the DPA-triggered lanthanide luminescence assay, we are exploring binary Tb3+ and Eu3+ complexes that act as DPA receptor sites with increased DPA affinity compared to Ln3+(aq) ions.19 To quantify the efficacy of these lanthanide-macrocycle platforms, we have developed and tested a binding affinity by competition (BAC) assay to determine DPA to binary complex binding constants. In contrast to the Benesi-Hildebrand method,20 the BAC assay does not break down when (1) binding constants are large (>109 M-1), (2) the system contains more than two components, or (3) changes in absorbance or luminescence are small.21,22 In our case, the (5) Murrel, W. G. The Bacterial Spore; Academic Press: New York, 1969; pp 215-273.. (6) Shafaat, H. S.; Ponce, A. Appl. Environ. Microbiol. 2006, 72 (10), 6808– 6814. (7) Yung, P. T.; Lester, E. D.; Bearman, G.; Ponce, A. Biotechnol. Bioeng. 2007, 98 (4), 864–871. (8) Hindle, A. A.; Hall, E. A. H. Analyst 1999, 124 (11), 1599–1604. (9) Rosen, D. L. Rev. Anal. Chem. 1999, 18 (1-2), 1–21. (10) Sacks, L. E. Appl. Environ. Microbiol. 1990, 56 (4), 1185–1187. (11) Jones, G.; Vullev, V. I. Photochem. Photobiol. Sci. 2002, 1 (12), 925–933. (12) Jones, G.; Vullev, V. I. J. Phys. Chem. A 2002, 106 (35), 8213–8222. (13) Sabbatini, N.; Guardigli, M.; Lehn, J. M. Coord. Chem. Rev. 1993, 123 (12), 201–228. (14) Balzani, V.; Decola, L.; Prodi, L.; Scandola, F. Pure Appl. Chem. 1990, 62 (8), 1457–1466. (15) Balzani, V. Pure Appl. Chem. 1990, 62 (6), 1099–1102. (16) Lehn, J. M. J. Inclusion Phenom. Macrocyclic Chem. 1988, 27 (1), 89–112. (17) Horrocks, W. D., Jr.; Sudnick, D. R. Acc. Chem. Res. 1981, 14, 384–392. (18) Horrocks, W. D., Jr. Prog. Inorg. Chem. 1984, 31, 1. (19) Cable, M. L.; Kirby, J. P.; Sorasaenee, K.; Gray, H. B.; Ponce, A. J. Am. Chem. Soc. 2007, 129 (6), 1474–1475. (20) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71 (8), 2703– 2707. (21) Wang, R.; Yu, Z. W. Acta Phys.-Chim. Sin. 2007, 23 (9), 1353–1359. (22) Yang, C.; Liu, L.; Mu, T. W.; Guo, Q. X. Anal. Sci. 2000, 16 (5), 537–539. 10.1021/ac800154d CCC: $40.75 2008 American Chemical Society Published on Web 06/26/2008
complex contains three components and operates in the high binding constant regime (>109 M-1), which is a requirement for sensitive detection of bacterial spores via DPA-triggered Tb3+/ Eu3+ luminescence. Here we report details of the BAC assay applied to determine the binding constant, Ka′, for DPA binding to the binary complex of Eu-(1,4,7,10-tetraazacyclododecane-1,7-diacetate) (i.e., Eu(DO2A)+), as shown in equilibrium 1. (Based on lifetime measurements of analogous Tb complexes,19 we assume that the remaining coordination sites of Eu(DPA)+ and Eu(DO2A)+ are occupied by water molecules.) Ka′
Eu(DO2A)+ + DPA2- H Eu(DO2A)(DPA)[Eu(DO2A)(DPA)-]eq Ka′ ) [Eu(DO2A)+]eq[DPA2-]eq
(1)
In order to determine Ka′, we performed a competitive binding experiment described by equilibrium 2, where the addition of excess Eu3+ to Eu(DO2A)(DPA)- results in the equimolar formation of the two species Eu(DO2A)+ and Eu(DPA)+. Kc
Eu(DO2A)(DPA)- + Eu3+ H Eu(DO2A)+ + Eu(DPA)+ [Eu(DO2A)+]eq[Eu(DPA)+]eq Kc ) [Eu(DO2A)(DPA)-]eq[Eu3+]eq
(2)
As Eu3+ is added, the shift in equilibrium (Eu(DO2A)(DPA)-) and Eu(DPA)+ concentrations are monitored via the ligand field sensitive 5D0 f 7F3 transition (λem ) 670-700 nm) using luminescence spectroscopy. A best fit of luminescence intensity titration data to a two-state thermodynamic model (see eq 10) yielded the competition equilibrium constant (Kc), which in conjunction with independent measurement of the Eu(DPA)+ formation constant (Ka) allowed calculation of the ternary complex formation constant (Ka′). EXPERIMENTAL SECTION Materials. DPA (dipicolinic acid, pyridine-2,6,-dicarboxylic acid) and EuCl3 · 6H2O were purchased from Sigma-Aldrich. Tetrabutylammonium hydroxide (10% in 2-propanol) was purchased from TCI America. Sodium acetate trihydrate and sodium hydroxide (50% in H2O) were purchased from Mallinckrodt Chemicals. All chemicals were used as received without further purification. All metal salts were greater than 99.99% pure. The 1,4,7,10-tetraazacyclododecane-1,7-diacetate (DO2A), which serves as the ligand platform, was prepared by hydrolysis of 1,4,7,10tetraazacyclododecane-1,7-di(tert-butyl acetate) purchased from Macrocyclics, as described by Huskens et al.,23 resulting in a white solid in 99.8% yield. DO2A · 2.80HCl · 0.85H2O. Anal. Calcd (found) for C12H24N4O4 · 2.80HCl · 0.85H2O (fw ) 405.57): C, 35.54 (35.54); H, 7.08 (6.72); N, 13.81 (13.25); Cl, 24.43 (25.10). Crystallization of Eu(DO2A)(DPA)-. Equimolar aliquots of EuCl3 · 6H2O (0.218 96 g, 0.59 mmol) and DO2A · 2.80HCl · 0.85H2O (0.243 60 g, 0.59 mmol) were dissolved in 2.00 mL of Nanopure H2O (18.2 MΩ resistance) using gentle heating and sonication. The pH of the solution was adjusted to ∼6 with tetrabutylammonium hydroxide (TBAOH, 10% in 2-propanol) added dropwise. An (23) Huskens, J. Inorg. Chem. 1997, 36 (7), 1495–1503.
equimolar amount of DPA (0.099 97 g, 0.59 mmol) was added to the solution, along with 1.00 mL of Nanopure H2O. The pH of the solution was adjusted to 8.0 with TBAOH, added dropwise. The solution was freeze-dried, and 18.0 mL of acetone was added to the resulting solid, which was sonicated and vortexed to solubilize as much of the ternary complex as possible. The mixture was centrifuged at 8000 rpm (25 °C) for 20 min, and the supernatant was decanted and filtered. Crystal formation was observed after 24 h at room temperature. Suitable crystals were utilized for X-ray diffraction studies, while the rest were dried over P2O5 under vacuum for 7 days and sent to Desert Analytics Transwest Geochem for elemental analysis. Anal. Calcd (found) for NC16H36 · EuC19H25N5O8 · 3.52H2O · 0.93C16H36NCl (fw ) 1168.8): C, 51.32 (51.33); H, 8.77 (8.00); N, 8.31 (8.49); Eu, 13.00 (12.95). Methods. Unless otherwise specified, all samples were prepared to a final volume of 4.00 mL in disposable acrylate cuvettes (Spectrocell), 1-cm path length, and were allowed to equilibrate for at least 7 days before analysis at 25 °C using a Fluorolog-3 Fluorescence spectrometer (Horiba Jobin-Yvon) operated in continuous mode with a xenon lamp (450 W). BAC Assay. Samples were prepared in triplicate using stock solutions of 400 µM Eu(DO2A)(DPA)- (prepared by solubilizing the TBA · Eu(DO2A)(DPA) crystals obtained previously), 4.00 mM Eu3+, and 400 µM Eu3+ with 200 mM sodium acetate (pH 7.5), such that the concentration of Eu(DO2A)(DPA)- was 1.0 µM and the concentration of free Eu3+ ranged from 1.0 nM to 1.0 mM. The use of X-ray quality, solvated TBA · Eu(DO2A)(DPA) crystals in this work demonstrates a major advantage, especially at low concentrations; the precise measure of the initial concentration of Eu(DO2A)(DPA)- could not be achieved without this important step. Association constants were calculated using the Curve Fitting Tool in Matlab with a chemical equilibrium model derived below. Computational Methods. We measured the luminescence intensity (Iobs) as a function of excess Eu3+ added ([Eu3+]xs). The titration data were fit using a two-state model (eq 3), assuming that only the two DPA-bound species contribute to the observed luminescence intensities (i.e., I{Eu(DO2A)(DPA)-} and I{Eu(DPA)+} . I{Eu(DO2A)+} and I{Eu3+}12). We define the concentrations of Eu(DO2A)(DPA)- and Eu(DPA)+ at equilibrium as [Eu(DO2A)(DPA)-]eq and [Eu(DPA)+]eq, respectively, and the total ternary complex concentration as [Eu(DO2A)(DPA)-]T. We define Imax and Imin as the maximum and minimum observed intensities, and c1 and c2 as the respective fractions of Eu(DO2A)(DPA)- and Eu(DPA)+ at equilibrium (i.e., c1 ) [Eu(DO2A)(DPA)-]eq/[Eu(DO2A)(DPA)-]T, c2 ) [Eu(DPA)+]eq/ [Eu(DO2A)(DPA)-]T, and c1 + c2 ) 1). Iobs ) c1Imax + c2Imin )+]eq
DPA ( Eu Eu )I DO2A DPA DPA ( Eu Eu )I DO2A DPA
) 1[
[
[
(
[
(
(
(
)-]T
)(
max +
)+]eq
)(
)-]T
min
(3)
To fit the data for Iobs versus [Eu3+]xs, we derive an expression for [Eu(DPA)+]eq in terms of [Eu3+]xs and Ka′ (eq 1). Toward this end, we consider the equilibrium of Eu3+ and DPA2- expressed in equilibrium 4. Analytical Chemistry, Vol. 80, No. 15, August 1, 2008
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Ka
Eu3+ + DPA2- H Eu(DPA)+ Ka )
[Eu(DPA)+]eq
[Eu3+]eq[DPA2-]eq
(4)
The value of Ka has been calculated, previously through potentiometric means24 and recently via a titrimetric method12 in similar conditions, to be 107.40 M-1. Since Kc is related to Ka′ by eq 5, and [Eu(DO2A)(DPA)-]eq and [Eu3+]eq can be expressed in terms of mass balance eqs 6 and 7, respectively, we obtain an expression for [Eu(DPA)+]eq in terms of Ka′, Ka, [Eu(DO2A)(DPA)-]T and [Eu3+]T. Kc )
Ka Ka′
)
2 [Eu(DPA)+]eq
[Eu(DO2A)(DPA)-]eq[Eu3+]eq
2 ) ([Eu(DPA)+]eq )⁄
[([Eu(DO2A)(DPA)-]T- [Eu(DPA)+]eq)([Eu3+]T [Eu(DO2A)(DPA)-]T - [Eu(DPA)+]eq)] (5) In eq 5, we assume that the DO2A2- ligand stays bound to Eu3+, given that log KGdDO2A )19.425 and log KEuDO2A ) 13.0.26 As a result, the total ternary complex concentration ([Eu(DO2A)(DPA)-]T) at equilibrium can be expressed as the sum of all Eu(DO2A)+-containing species (eq 6).
[Eu(DO2A)(DPA)-]T ) [Eu(DO2A)(DPA)-]eq + [Eu(DO2A)+]eq
(6)
) [Eu(DO2A)(DPA)-]eq + [Eu(DPA)+]eq
The total Eu3+ concentration ([Eu3+]T) at equilibrium is defined as the sum of all Eu3+-containing equilibrated species (eq 7).
[Eu3+]T ) [Eu3+]eq + [Eu(DO2A)(DPA)-]eq + [Eu(DO2A)+]eq + [Eu(DPA)+]eq 3+ ) [Eu ]eq + [Eu(DO2A)(DPA)-]eq + 2[Eu(DPA)+]eq (7) Solving eq 5 for [Eu(DPA)+]eq, we obtain eq 8. [Eu(DPA)+]eq )
[Eu3+]T +
({
-] }2 [Eu3+]T - 2(1 - Ka′K-1 [[ a ) Eu(DO2A)(DPA) T ′ -1 ′ -1 ([ -] )2 +4Ka Ka (1 - Ka Ka ) Eu(DO2A)(DPA) T
2(1 - Ka′K-1 a )
)
1⁄2
(8)
The total concentration of the ternary complex, [Eu(DO2A)(DPA)-]T, is experimentally fixed (in this case 1.0 µM), and the total Eu3+ concentration ([Eu3+]T) is the sum of this initial ternary complex concentration and any excess Eu3+ added ([Eu]XS) as shown in eq 9.
[Eu3+]T ) [Eu(DO2A)(DPA)-]T + [Eu3+]xs
(9)
Substituting the expression for [Eu(DPA)+]eq from eq 8 into eq 3, we obtain the BAC assay two-state model expression used to calculate Ka′. Iobs ) (1 - (2AB)-1{A + [Eu3+]XS + ({A + [Eu3+]XS - 2AB}2 + 4A(1 - A)B2)1⁄2})Imax + ((2AB)-1{A + [Eu3+]XS + ({A + [Eu3+]XS - 2AB}2 + 4A(1 - A)B2)1⁄2})Imin (10) where A ) [Eu(DO2A)(DPA)- ]T and B ) 1 - Ka′ · Ka-1. RESULTS Characterization of Eu(DO2A)(DPA)-. The difference in the emission spectra (5D0 f 7F3 transition) of Eu(DPA)+ and Eu(DO2A)(DPA)- is striking, and can be employed to monitor the equilibrium concentrations during competition experiments. Full emission and excitation spectra are available in Supporting Information. The crystal structure confirms hexadentate coordination of the macrocyclic DO2A2- ligand to the central Eu3+ ion, with the three remaining adjacent sites occupied by the tridentate DPA2- species (Figure 1). BAC Assay. With the association constant of Eu3+ to DPA2known (see Supporting Information) and the spectroscopic differences between Eu(DO2A)(DPA)- and Eu(DPA)+ confirmed, we were able to apply the BAC assay to calculate the affinity of the lanthanide-macrocycle platform Eu(DO2A)+ for DPA2- in this system. Using the solvated TBA · Eu(DO2A)(DPA) crystals to achieve an optimized 1:1:1 ratio of Eu/DO2A/DPA, we were able to perform the titration over 6 orders of magnitude, with added [Eu3+]XS ranging from 1.0 nM to 1.0 mM. The shift from ternary to binary complex was easily observed via luminescence spectroscopy in this range (Figure 2), and the ligand field sensitive 5D0 f 7F3 peak was integrated to produce a titration curve (Figure 3). Fitting each titration curve to the BAC assay two-state model (eq 10), we calculated an average value of 0.154 for Kc, yielding an association constant log Ka′ ) 8.21 ± 0.02. Titration of free Eu3+ in buffer over the same concentration range resulted in negligible intensity increase, confirming that the intensity change observed is due to the transition from ternary to monoDPA complex. DISCUSSION The BAC assay detailed here can be employed to quantify ternary lanthanide complex formation under conditions where the
Figure 1. Thermal ellipsoid plot of the Eu(DO2A)(DPA)- ternary complex with 50% probability. Hydrogens omitted for clarity. 5752
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(24) Grenthe, I. J. Am. Chem. Soc. 1961, 83 (2), 360–364. (25) Kim, W. D.; Hrncir, D. C.; Keifer, G. E.; Sherry, A. D. Inorg. Chem. 1995, 34 (8), 2225–2232. (26) Chang, C. A.; Chen, Y. H.; Chen, H. Y.; Shieh, F. K. J. Chem. Soc., Dalton Trans. 1998, (19), 3243–3248.
Figure 2. Emission spectra of the ligand field sensitive 5D0 f 7F3 transition for Eu3+ of the BAC assay. The shift from the ternary Eu(DO2A)(DPA)species (solid) to the mono-Eu(DPA)+ species (dashed) as [Eu3+]XS is increased up to 1.0 mM is easily discernible. Concentration of ternary complex is 1.0 µM, pH 7.5. Excitation at 278 nm.
Figure 3. BAC assay titration to calculate the association constant (Ka′) of Eu(DO2A)+ and DPA2-. Initial concentration of Eu(DO2A)(DPA)was 1.0 µM, pH 7.5.
lanthanide-macrocycle platform is stable and the concentrations of the two states of the receptor sitesbound and vacantscan be measured. In our case, the binary and ternary complexes of interest are spectroscopically resolvable. The BAC assay is especially useful in the high binding regimes, where direct measures of ligand binding, such as Benesi-Hildebrand method, break down. In addition, the BAC assay is valid over 6 orders of magnitude in concentration. We believe that this luminescence method will find many applications, including but not limited to
immunoassays,27–29 DNA hybridization assays,30,31 protein analysis,32 high-throughputscreeningandimaging,33,34 anionsensing,35–37
(27) Hemmila, I.; Dakubu, S.; Mukkala, V. M.; Siitari, H.; Lovgren, T. Anal. Biochem. 1984, 137 (2), 335–343. (28) Diamandis, E. P. Analyst 1992, 117 (12), 1879–1884. (29) Meyer, J.; Karst, U. Analyst 2000, 125 (9), 1537–1538. (30) Yuan, J. L.; Wang, G. L. TrAC-Trends Anal. Chem. 2006, 25 (5), 490–500. (31) Kitamura, Y.; Ihara, T.; Tsujinura, Y.; Osawa, Y.; Tazaki, M.; Jyo, A. Anal. Biochem. 2006, 359 (2), 259–261.
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pH determination,38 and targeted detection assays for specific chromophores.39–42 Surprisingly, the calculated association constant (Ka′) of Eu(DO2A)+ and DPA2- is nearly 1 order of magnitude greater than that of the Eu3+ cation alone at the same pH (log Ka ) 7.40 ± 0.03). We have observed and reported a similar trend in the analogous Tb3+ system, though in both cases, such a result does not follow predictions based on total charges of the binding species. Specifically, upon addition of the DO2A ligand, the dipicolinate receptor site decreases from a net 3+ charge of the lanthanide alone to a binary complex with a total charge of only 1+. We postulate that the lanthanide-macrocycle platform helps to maximize the positive surface area of the Tb3+ binding site through addition of the macrocycle amine hydrogens, which allows for greater compatibility with the negative surface of the dipicolinate moiety. We emphasize that the BAC assay allows for the unambiguous measure of relative stability, to guide us toward a superior sensor for bacterial spores, including those produced by the causative agent of anthrax. CONCLUSION The BAC assay was developed and successfully applied to determine binding affinities of DPA2- for Eu(DO2A)+ in the high (32) Santos, M.; Roy, B. C.; Goicoechea, C.; Campiglia, A. D.; Malik, S. J. Am. Chem. Soc. 2004, 126 (34), 10738–10745. (33) Papanastasiodiamandi, A.; Christopoulos, T. K.; Diamandis, E. P. Clin. Chem. 1992, 38 (4), 545–548. (34) Lin, Z. H.; Wu, M. Chirality 2005, 17 (8), 464–469. (35) Beer, P. D.; Gale, P. A. Angew. Chem., Int. Ed. 2001, 40 (3), 486–516. (36) Gale, P. A.; Quesada, R. Coord. Chem. Rev. 2006, 250 (23-24), 3219– 3244. (37) Snowden, T. S.; Anslyn, E. V. Curr. Opin. Chem. Biol. 1999, 3 (6), 740– 746. (38) Gunnlaugnsson, T.; Mac Donaill, D. A.; Parker, D. J. Am. Chem. Soc. 2001, 123 (51), 12866–12876. (39) Zhu, R. H.; Kok, W. T. Anal. Chem. 1997, 69 (19), 4010–4016. (40) Arnaud, N.; Georges, J. Analyst 1999, 124, 1075–1078. (41) Mortellaro, M. A.; Nocera, D. G. J. Am. Chem. Soc. 1996, 118 (31), 7414– 7415. (42) Lianidou, E. S.; Ioannou, P. C.; Polydorou, C. K.; Efstathiou, C. E. Anal. Chim. Acta 1996, 320 (1), 107–114.
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binding constant regime (>109 M-1). This method serves as a diagnostic tool in our efforts to improve the Tb-DPA luminescence bacterial spore assay by employing lanthanide-macrocycle platforms with increased DPA2- affinity. The BAC assay for DPA to binary complex binding affinity determination is especially useful in the high binding constant regime, where direct analyte titration approaches are not feasible due to experimental limitations. This technique can be applied to any receptor site where the binary and ternary complexes are spectroscopically different and can be implemented in a variety of conditions such as those expected in environmental samples. We plan to apply the BAC assay to test various ligands, anions, and cations in our quest for improved bacterial spore receptor sites. ACKNOWLEDGMENT The authors thank Larry Henling and Mike Day for assistance. M.L.C. acknowledges support from the National Defense Science and Engineering Graduate (NDSEG) Fellowship Program. D.J.L. was supported by the Amgen Fellowship Program. Work at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration was supported by NASA’s Astrobiology and Planetary Protection Programs and Department of Homeland Security’s Chemical and Biological Research & Development Program. Work at the Beckman Institute was supported by NSF and the Arnold and Mabel Beckman Foundation. SUPPORTING INFORMATION AVAILABLE Crystallographic data (CIF), full excitation and emission spectra, and pH dependence data. This material is available free of charge via the Internet at http://pubs.acs.org.
Received for review January 22, 2008. Accepted April 18, 2008. AC800154D
