Conjugation of Au11 cluster with Cys-rich peptides containing the α-domain of metallothionein

Abstract

The stoichiometrically controlled complexation of abiological inorganic clusters with proteins is a challenging task that enables us to combine the functions of inorganic clusters with those of biological systems on the basis of chemical characterisation. In this study, we synthesised a 1:1 conjugate between Au₁₁(PPh₃)₈Cl₃ (Au11) and CGMTIIα, where Au11 is the smallest cluster, which is situated on the boundary of molecules and nanoparticles, having discrete electronic states and exhibiting Coulomb blockade; on the other hand, CGMTIIα is a twelve-cysteine (Cys)-containing peptide consisting of the α-domain of rat liver metallothionein II (MTIIα)—a heavy metal-binding protein—and an addendum, cysteine–glycine (CG). The Cys residue of the N-terminus is used as a site for elongation by the native chemical ligation (NCL) method. Titration experiments monitored by circular dichroism spectroscopy indicate that CGMTIIα reacts with Au11 in a ratio of 1:1. The product exhibits a negative Cotton effect at 222 nm, indicating the formation of a short a-helix. The composition of this hybrid module is quantified as Au₁₁(PPh₃)₄(S-Cys)₆ by transmission electron microscopy (core size observation), energy-dispersive X-ray spectroscopy (elemental analysis), Ellman test (uncoordinated Cys residues), and high-performance liquid chromatography (quantification of eliminated PPh₃). These results, as well as those obtained from the simulations of Au 4f X-ray photoelectron spectra, suggest that the Au cluster of this product is Au₁₁(PPh₃)₄(S-Cys)₆. We also prepared F3F3–CGMTIIα, where F3F3 is a zinc finger peptide with two domains, in order to demonstrate the applicability of the NCL method to the elongation of CGMTIIα. F3F3–CGMTIIα also reacts with Au11 to yield a 1:1 conjugate without interfering in the folding of the zinc finger.