Spectroscopic Studies on the Designed Metal-Binding Sites of the 43C9 Single Chain Antibody

In an effort to expand the catalytic repertoire of antibodies to encompass metal ion-assisted reactions, three classes of metal ion-binding sites were designed within the antigen-binding site of catalytic single-chain antibody (SCA) 43C9 and characterized by a variety of spectroscopic techniques. With structural motifs of metalloenzymes as prototypes, computer modeling techniques were used to design these sites. The affinities of each class of metal ion-binding sites for a variety of divalent metal ions were determined by fluorescence quenching techniques. One class of binding sites, consisting of His residues at positions L32, L34, and L91, bound Zn(II) with a K_Dvalue of 3.3 ± 0.8 μM; however, the affinity for the inducing antigen was decreased by at least 10⁴ relative to that of the wild-type in the absence of Zn(II). The second class of metal ion-binding sites, which consisted of His residues at positions H33, H35, and H95, possessed greater than 100-fold selectivity for Zn(II) over any other divalent metal ion tested and bound this ion with K_Dvalues of 1.5—3.7 μM. The third class of metal ion-binding sites utilized His residues at positions L91 and L96 and, in some cases, H95. This class was selective for Cu(II) over Zn(II), binding the former with K_D values of 0.5—2.1 μM and the latter with K_Dvalues of 10–40 μM. Continuous-wave EPR studies of Cu(II) bound to this class of mutants verified the results of the fluorescence quenching assays; Cu(II) binding resulted in EPR signals that were well approximated by a simulation using the parameters of A_||= 166.0G, A_⊥ = 6.0G, g_||= 2.19, and g_⊥= 2.05. Furthermore, pulsed EPR experiments (ESEEM) demonstrated that (1) a low-affinity Cu(II) site (K_D≥100 μM) consisting of a single His residue existed in the wild-type SCA, (2) two His residues acted as Cu(II) ligands in the R-L96-H single mutant, and (3) three His residues acted as Cu(II) ligands in the R-L96-H, Y-H95-H double mutant. These results are consistent with the original computational design. In addition, UV—vis studies suggested a rare Tyr to Cu(II), ligand-to-metal charge transfer band at 490 nm (∊ = 55 M^-1 cm^-1) that arose from coordination of the Tyr H95 side chain to the bound Cu(II). As expected, this band was absent in the Cu(II)-bound form of the R-L96-H, Y-H95-F double mutant SCA. Finally, the R-L96-H mutant was shown to simultaneously bind metal and p-nitrophenol.