Insights into Intramolecular Trp and His Side-Chain Orientation and Stereospecific pi Interactions Surrounding Metal Centers: An Investigation Using Protein Metal-Site Mimicry in Solution

Metal-binding scaffolds incorporating a Trp/His-paired epitope are instrumental in giving novel insights into the physicochemical basis of functional and mechanistic versatility conferred by the Trp-His interplay at a metal site. Herein, by coupling bio-metal site mimicry and H-1 and C-13 NMR spectroscopy experiments, modular constructs EDTA-(L-Trp, L-His) (EWH; EDTA=ethylenediamino tetraacetic acid) and DTPA-(L-Trp, L-His) (DWH; DTPA = diethylenetriamino pentaacetic acid) were employed to dissect the static and transient physicochemical properties of hydrophobic/hydrophilic aromatic interactive modes surrounding biometal centers. The binding feature and identities of the stoichiometric metal-bound complexes in solution were investigated by using H-1 and C-13 NMR spectroscopy, which facilitated a cross-validation of the carboxylate, amide oxygen, and tertiary amino groups as the primary ligands and indole as the secondary ligand, with the imidazole (1m) N3 nitrogen being weakly bound to metals such as Ca2+ owing to a multi-valency effect. Surrounding the metal centers, the stereospecific orientation of aromatic rings in the diastereoisomerism is interpreted with the Ca2+-EWH complex. With respect to perturbed Trp side-chain rotamer heterogeneity, drastically restricted Trp side-chain flexibility and thus a dynamically constrained rotamer interconversion due to pi interactions is evident from the site-selective C-13 NMR spectroscopic signal broadening of the Trp indolyl C3 atom. Furthermore, effects of Trp side-chain fluctuation on indole/Im orientation were the subject of a 2D NMR spectroscopy study by using the Ca2+-bound state; a C-H2(indolyl)/C-H5(Im(+)) connectivity observed in the NOESY spectra captured direct evidence that the N-H1 of the Ca2+-Im(+) unit interacted with the pyrrole ring of the indole unit in Ca2+-bound EWH but not in DWH, which is assignable to a moderately static, anomalous, T-shaped, interplanar pi(+)-pi stacking alignment. Nevertheless, a comparative C-13 NMR spectroscopy study of the two homologous scaffolds revealed that the overall response of the indole unit arises predominantly from global attractions between the indole ring and the entire positively charged first coordination sphere. The study thus demonstrates the coordination-sphere/geometry dependence of the Trp/His side-chain interplay, and established that pi interactions allow C-13 NMR spectroscopy to offer a new window for investigating Trp rotamer heterogeneity near metal-binding centers.