Conjugation of Au11 cluster with Cys-rich peptides containing the alpha-domain of metallothionein.
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 CGMTIIalpha, 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, CGMTIIalpha is a twelve-cysteine (Cys)-containing peptide consisting of the alpha-domain of rat liver metallothionein II (MTIIalpha)--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 CGMTIIalpha 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-CGMTIIalpha, where F3F3 is a zinc finger peptide with two domains, in order to demonstrate the applicability of the NCL method to the elongation of CGMTIIalpha. F3F3-CGMTIIalpha also reacts with Au11 to yield a 1:1 conjugate without interfering in the folding of the zinc finger.
Department of Chemistry, Faculty of Science, Tokyo University of Science, 1-3, Kagurazaka, Shinjuku-ku 162-0826, Tokyo, Japan.
Shinya
S
Ariyasu
Akira
A
Onoda
Ryota
R
Sakamoto
Takeshi
T
Yamamura
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D008668:Metallothionein / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009942:Organometallic Compounds / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D051381:Rats; D016335:Zinc Fingers
D009942:Organometallic Compounds; D010455:Peptides; D006046:Gold; D008668:Metallothionein; D003545:Cysteine