PROTEIN SURFACE RECOGNITION AND COVALENT BINDING BY CHROMIUM NITRILOTRIACETATE COMPLEXES - ELUCIDATION USING NMR AND CD SPECTROSCOPIES

New approaches to delineate sites of noncovalent and covalent binding by small metal complexes on a protein surface are described. The complexes employed are based on the chromium(III) nitrilotriacetate framework substituted with amino acid side chain groups. The protein examined is the well-characterized hen egg white lysozyme. Noncovalent binding location and mode have been studied by the technique of nuclear Overhauser effect quenching, along with paramagnetic relaxation experiments. These methods allow the elucidation of two major and two minor binding sites on the protein surface. The binding sites of greater intensity revolve around His-15 and active cleft residues Ala-107, Ile-98, and Trp-63 C2H, respectively. A comparison of the binding interaction as a function of the metal complex chelate ring substitutions suggests that the binding mode is modified by the side chain moiety; in particular, an aromatic-aromatic interaction between bound metal complex and aromatic residues on the protein surface is inferred. In addition to noncovalent interactions, the covalent labeling of coordinating protein side chains is examined. This is studied using circular dichroism to monitor the induction of vicinal dissymmetry in a symmetric metal complex through coordination of an amini acid side chain. These experiments indicate that aspartic acid residues near the sites of noncovalent association are labeled. As the sites of covalent labeling correlate well with the sites of noncovalent interaction, it is likely that the noncovalent binding influences the sites of covalent attachment.