Europium Luminescence of EF-Hand Helix−Turn−Helix Chimeras:  Impact of pH and DNA-Binding on Europium Coordination

A series of Eu(III) metallopeptides, designed on the basis of the structural similarity of the helix−turn−helix and EF-hand motifs, have been studied by Eu(III) 7F0 → 5D0 excitation spectroscopy. The impact of EF-hand ligand set differences on the hydration number and Eu(III) coordination environment are compared among the peptides. The conditional binding affinities were determined by Eu titration (P3, log K a = 6.0 ± 0.4; P3W, log K a = 5.9 ± 0.2; P5b, log K a = 5.3 ± 0.1). Two similar coordination environments occur in each case, consistent with structural flexibility about the metal site. The coordination environments are consistent with 8- or 9-coordinate Eu(III), including six peptide-based ligands and two to three water molecules (P3, q = 1.9 ± 0.2; P3W, q = 2.3 ± 0.2; P4a, q = 1.9 ± 0.3; P5b, q = 2.6 ± 0.2). The Eu(III) 7F0 → 5D0 excitation spectra are pH-dependent, as reported for several EF-hand proteins (oncomodulin, parvalbumin). A higher energy transition occurs at pH > 6, and has been assigned to deprotonation of coordinated water. The pK a leading to this new transition is dependent on Eu(III) Lewis acidity, which varies with the inner and outer sphere ligand set. The noncoordinating ninth position of the Eu-binding loop, which is poised to make second-sphere contacts to the coordinated water, stabilizes the deprotonated form of the coordinated solvent more effectively when it is Thr (P5b) than Asp (P3W). Upon DNA-binding by the metallopeptides, the pK a of the pH-dependent peak increases, but no new DNA-dependent transitions are observed. This indicates no DNA-based Eu(III) ligands are introduced, such as phosphate oxygen atoms of the DNA backbone. The hydration number decreases in the presence of DNA (P3W + DNA, q = 1.9 ± 0.2; P5b + DNA, q = 1.7 ± 0.2), indicating that DNA-binding by the metallopeptides organizes rather than compromises the Eu-binding site within the peptide.