Metal‐Ion‐Regulated Miniature DNA‐Binding Proteins Based on GCN4 and Non‐native Regulation Sites

Abstract

The design of artificial peptide dimers containing polypyridine switching domains, for which metal-ion coordination is shown to regulate DNA binding, is reported. Short peptides, based on the basic domain of the GCN4 transcription factor (GCN4bd), dimerised with either 2,2'-bipyridine (bipy(GCN4bd) 2 ) or 2,2':6',2''-terpyridine (terpy(GCN4bd) 2 ) linker units, undergo a conformational rearrangement on Cu II and Zn II coordination. Depending on the linker substitution pattern, this is proposed to alter the relative alignment of the two peptide moieties, and in turn regulate DNA binding. Circular dichroism and UV-visible spectroscopy reveal that Cu II and Zn II coordination promotes binding to DNA containing the CRE target site, but to a differing and opposite degree for the two linkers, and that the metal-ion affinity for terpy(GCN4bd) 2 is enhanced in the presence of CRE DNA. Binding to DNA containing the shorter AP1 target site, which lacks a single nucleobase pair compared to CRE, as well as half-CRE, which contains only half of the CRE target site, was also investigated. Cu II and Zn II coordination to terpy(GCN4bd) 2 promotes binding to AP1 DNA, and to a lesser extent half-CRE DNA. Whereas, bipy(GCN4bd) 2 , for which interpeptide distances are largely independent of metal-ion coordination and less suitable for binding to these shorter sites, displays allosteric ineffective behaviour in these cases. These findings for the first time demonstrate that biomolecular recognition, and specifically sequence-selective DNA binding, can be controlled by metal-ion coordination to designed switching units, non-native regulation sites, in artificial biomolecules. We believe that in the future these could find a wide range of applications in biotechnology.

Tunable helicity, stability and DNA-binding properties of short peptides with hybrid metal coordination motifs

“…In this study, we exploited this feature to demonstrate the ability of HCM-bearing peptides to recognize and bind a biological target. Starting with the pioneering work by Kim, 54,61 several synthetic strategies have been developed for the dimerization of bZIP peptides, 55,62-64 including disulfide bonding, 54 metal coordination, 65-68 and crosslinking with metal-tunable 69 or photo-switchable linkers. 70-72 These studies have collectively shown the importance of the dimeric organization of the basic domains as well as their proper orientation for DNA binding and recognition.…”

Sequence‐Specific DNA Recognition with Designed Peptides

“…(g) Mascareñas' cis/trans azobenzene photoswitch linker . (h) Peacock's metal switch linker . (i) Peacock's photocontrolled anthracene dimer …”

“…This linker should undergo a conformational change upon metal ion coordination, which in turn should affect the relative orientation of the basic regions and influence the DNA binding of the dimer. CD and UV/Vis spectroscopy demonstrated that coordination of this metal‐chelating GCN4 dimer to Cu II or Zn II ions promoted DNA binding (Figure h) …”

Ditopic Aza-Scorpiand Ligands Interact Selectively with ds-RNA and Modulate the Interaction upon Formation of Zn2+ Complexes

“…Among the triggering stimuli, metal coordination is one of the most challenging strategies due to the abundance of metal ions in cells that can occupy the coordination sites of the synthetic systems and hamper the envisaged DNA interaction process. In this regard, Peacok’s team reported two short peptides connected by a bipyridine or a terpyridine unit that undergo conformational changes upon Zn 2+ or Cu 2+ coordination, prompting their interaction with double-stranded DNA (ds-DNA) [ 9 ]. An interesting system described by Learte-Aymami et al is composed of a peptide moiety containing histidine units, which is unable to bind DNA because of the unstructured nature in solution, but Pd 2+ coordination to the nitrogen atoms of the histidines promotes its folding, adopting a helical conformation with a concomitant high ds-DNA binding [ 10 ].…”