SPECTROSCOPIC STUDIES ON THE DESIGNED METAL-BINDING SITES OF THE 43C9SINGLE-CHAIN ANTIBODY

In an effort to expand the,catalytic repertoire of antibodies to encom pass metal ion-assisted reactions, three classes of metal ion-binding sites were designed within the antigen-binding site of catalytic singl e-chain antibody (SCA) 43C9 and characterized by a variety of spectros copic techniques. With structural motifs of metalloenzymes as prototyp es, computer modeling techniques were used to design these sites. The affinities of each class of metal ion-binding sites for a variety of d ivalent metal ions were determined by fluorescence quenching technique s. One class of binding sires, consisting of His residues at positions L32, L34, and L91, bound Zn(II) with a K-D value of 3.3 +/- 0.8 mu M; however, the affinity for the inducing antigen was decreased by at le ast 10(4) 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 re sidues at positions H33, H35, and H95, possessed greater than 100-fold selectivity for Zn(II) over any other divalent metal ion tested and b ound this ion with K-D values of 1.5-3.7 mu M. The third class of meta l 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 mu M and the latter wit h K-D values of 10-40 mu M. Continuous-wave EPR studies of Cu(II) boun d to this class of mutants verified the results of the fluorescence qu enching assays; Cu(II) binding resulted in EPR signals that were well approximated by a simulation using the parameters of A(parallel to) = 166.0G, A(perpendicular to) = 6.0G, g(parallel to) = 2.19, and g(perpe ndicular to) = 2.05. Furthermore, pulsed EPR experiments (ESEEM) demon strated that (1) a low-affinity Cu(II) site (K-D greater than or equal to 100 mu M) consisting of a single His residue existed in the wild-t ype SCA, (2) two His residues acted as Cu(II) ligands in the R-L96-H s ingle mutant, and (3) three His residues acted as Cu(II) ligands in th e R-L96-H, Y-H95-H double mutant. These results are consistent with th e original computational design. in addition, UV-vis studies suggested a rare Tyr to Cu(II), ligand-to-metal charge transfer band at 490 nm (epsilon = 55 M(-1) cm(-1)) that arose from coordination of the Tyr H9 5 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. Fina lly, the R-L96-H mutant was shown to simultaneously bind metal and p-n itrophenol.