Figure 1. The chemical structures of CP1, CP2, and CP3: CP1 contains 4 Glu carboxylate groups spaced adjacent to one another, imparting amphipathic in the cyclooctapeptide, CP2 contains 4 Glu carboxylate groups spaced further apart by alternating Glu residues with hydrophobic Leu residues, and CP3 contains 2 Glu carboxylate and 2 Cys thiolate groups spaced further apart by alternating Leu residues. The side chain coordinating atoms and the intrinsic fluorophore Trp are shown in bold.

Figure 2. ITC data curve of CP2 (a), and CP3 (b), following titration with Hg²⁺. Raw ITC titration data of 1.34 mL of CP2 (0.14 mM) and CP3 (0.15 mM) with 7 and 3 mM Hg(ClO₄)₂, respectively (top panels). Binding isotherms (bottom panels) are derived from the data in the corresponding top panels following correction for dilution and mixing effects.

Figure 3. ITC data curve of CP2 (a), and CP3 (b), following titration with Pb²⁺. Raw ITC titration data of 1.34 mL of CP2 (0.14 mM) and CP3 (0.15 mM) with 3 mM Pb(ClO₄)₂, (top panels). Binding isotherms (bottom panels) are derived from the data in the corresponding top panels following correction for dilution and mixing effects.

Figure 4. ITC data curve of CP2 (a), and CP3 (b), following titration with Cd²⁺ at 26 °C. Raw ITC titration data of 1.34 mL of CP2 and CP3 (0.15 mM) with 3 mM Cd(ClO₄)₂ at 33 injections of 6 μL each (top panels).

Figure 5. (a) Fluorescence emission spectrum of 10 μM CP3 in the absence and presence of increasing concentrations of Hg²⁺ (2.5, 5, 7.5, 10, 20, 40, 60, 110 μM), and (b) Fluorescence quenching of 10 μM CP1–3 (λ_em = 350 nm) in the presence of increasing concentrations of Hg²⁺. Measured at 25 °C with λ_ex = 281 nm.

Figure 6. (a) Fluorescence emission spectrum of 10 μM CP3 in the absence and presence of increasing concentrations of Pb²⁺ (10, 20, 30, 40, 50, 60, 80, 100, 150 μM), and (b) Fluorescence quenching of 10 μM CP1–3 (λ_em = 350 nm) in the presence of increasing concentrations of Pb²⁺. Measured at 25 °C with λ_ex = 281 nm.

Figure 7. Stern–Volmer plot for Hg²⁺ (a), and Pb²⁺ (b) quenching of 10 μM CP1–3. (a) Exponential transformation of Hg²⁺ quenching of CP1 and CP2 yielded the least square regression lines, y = 0.026x + 0.090, and y = 0.016x − 0.030, respectively, with coefficients of determination, r² > 0.99. Line of best fit analysis of Hg²⁺ quenching of CP3 yielded the linear regression line, y = 0.2135x + 1, where r² > 0.99. (b) Log transformation of Pb²⁺ quenching of CP1 and CP2 yielded the least square regression lines, y = 2.31x − 0.81, and y = 2.20x − 0.79, respectively, with coefficients of determination, r² > 0.97. Line of best fit analysis of Pb²⁺ quenching of CP3 yielded the linear regression line, y = 0.0155x + 1, where r² > 0.99.

Thermodynamic parameters of metal-ion binding to cyclooctapeptides^a