Biometal binding-site mimicry with modular, hetero-bifunctionally modified architecture encompassing a Trp/His motif: Insights into spatiotemporal noncovalent interactions from a comparative spectroscopic study

Abstract

Metal-site Trp/His interactions are crucial to diverse metalloprotein functions. This paper presents a study using metal-motif mimicry to capture and dissect the static and transient components of physicochemical properties underlying the Trp/His aromatic side-chain noncovalent interactions across the first- and second-coordination spheres of biometal ions. Modular biomimetic constructs, EDTA-(L-Trp, L-His) or EWH and DTPA-(L-Trp, L-His) or DWH, featuring a function-significant Trp/His pair, enabled extracting the putative hydrophobic/hydrophilic aromatic interactions surrounding metal centers. Fluorescence, circular dichroism (CD) spectroscopic titrations and ESI mass spectrometry demonstrated that both the constructs stoichiometrically bind to Ca²⁺, Co²⁺, Cu²⁺, Ni²⁺, Mn²⁺, Zn²⁺, Cd²⁺, and Fe²⁺, and such binding was strongly coupled to stereospecific side-chain structure reorientations of the Trp indole and His imidazole rings. A mechanistic dichotomy corresponding to the participation of the indole unit in the binding event was revealed by a scaffold-platform correlation of steady-state fluorescence-response landscape, illuminating that secondary-coordination-sphere ligand cation-π interactions were immediately followed by subsequent transient physicochemical processes including through-space energy transfer, charge transfer and/or electron transfer, depending on the type of metals. The fluorescence quenching of Trp side chain by 3d metal ions can be ascribed to through-space d-π interactions. While the fluorescence titration was capable of illuminating a two-component energetic model, clean isosbestic/isodichroic points in the CD titration spectra indicated that the metallo-constructs, such as Cu²⁺-EWH complex, fold thermodynamically by means of a two-state equilibrium. Further, the metal-ion dependence of Trp conformational variation in the modular architecture of metal-bound scaffolds was evidenced unambiguously by the CD spectra and supported by MMFF calculations; both were capable of distinguishing between the coordination geometry and the preference for metal binding mode. The study thus helps understand how aromatic rings around metal-sites have unique capabilities through the control of the spatiotemporal distribution of noncovalent interaction elements to achieve diverse chemical functionality.