Sequence-Specific DNA Binding by Noncovalent Peptide–Azocyclodextrin Dimer Complex as a Suitable Model for Conformational Fuzziness

Abstract

Transcription factors are proteins lying at the endpoint of signaling pathways that control the complex process of DNA transcription. Typically, they are structurally disordered in the inactive state, but in response to an external stimulus, like a suitable ligand, they change their conformation, thereby activating DNA transcription in a spatiotemporal fashion. The observed disorder or fuzziness is functionally beneficial because it can add adaptability, versatility, and reversibility to the interaction. In this context, mimetics of the basic region of the GCN4 transcription factor (Tf) and their interaction with dsDNA sequences would be suitable models to explore the concept of conformational fuzziness experimentally. Herein, we present the first example of a system that mimics the DNA sequence-specific recognition by the GCN4 Tf through the formation of a non- covalent tetra-component complex: peptide–azoβ-CyD(dimer)–peptide–DNA. The non-covalent complex is constructed on the one hand by a 30 amino acid peptide corresponding to the basic region of GCN4 and functionalized with an adamantane moiety, and on the other hand an allosteric receptor, the azoCyDdimer, that has an azobenzene linker connecting two β-cyclodextrin units. The azoCyDdimer responds to light stimulus, existing as two photo-states: the first thermodynamically stable with an E:Z isomer ratio of 95:5 and the second obtained after irradiation with ultraviolet light, resulting in a photostationary state with a 60:40 E:Z ratio. Through electrophoretic shift assays and circular dichroism spectroscopy, we demonstrate that the E isomer is responsible for dimerization and recognition. The formation of the non-covalent tetra component complex occurs in the presence of the GCN4 cognate dsDNA sequence (′5-..ATGA cg TCAT..-3′) but not with (′5-..ATGA c TCAT..-3′) that differs in only one spacing nucleotide. Thus, we demonstrated that the tetra-component complex is formed in a specific manner that depends on the geometry of the ligand, the peptide length, and the ds DNA sequence. We hypothesized that the mechanism of interaction is sequential, and it can be described by the polymorphism model of static fuzziness. We argue that chemically modified peptides of the GCN4 Tf are suitable minimalist experimental models to investigate conformational fuzziness in protein–DNA interactions.

Possible functions of the conserved peptide encoded by the RNA-precursor of miR-156a in plants of the familyBrassicaceae

“…A significant number of these proteins interact with chromatin (Peng et al, 2015; Wang et al, 2016). Recently, the interaction of a DNA-binding peptide of the transcription factor GCN4 with model DNA was studied (Quirolo et al, 2019). Importantly, a monomeric peptide, based on GCN4 sequence and including 34-amino acid residues, was not able to bind to a specific dsDNA.…”

“…7). In the case of GCN4 peptide, which is strongly disordered in a free state, its folding complex with DNA resulted in more alpha helices (Quirolo et al, 2019). However, miPEP156a has an increase in the number of beta structures when it interacts with DNA (Table 2).…”

Photocontrolled DNA minor groove interactions of imidazole/pyrrole polyamides

“…In a previous study, the same azobenzene species incorporated in a hairpin peptide reached a photostationary state of 85% Z-isomer [35]. The photostationary state depends on the nature of the azobenzene, but also on the concentration, as the azobenzene is known to form supramolecular assemblies at high con- centration, compromising photoconversion [11,12]. When the concentration of polyamide P1 was reduced to 0.8 mM, 80% of the Z-isomer were obtained after 15 min of irradiation ( Figure 2).…”

“…Along this line, it is attractive to incorporate a molecular switch for the selective activation of an initially inactive substance, once located at the DNA target, with an external stimulus. Azobenzenes are photoswitchable molecules capable of generating significant structural changes in peptides or small molecules, thereby controlling their geometry and functionality upon irradiation [10][11][12][13]. While isomerization of the E-to the Z-form usually takes place at 350 nm irradiation (π-π* transition), the reverse is photochemically induced at 450 nm (n-π* transition) or thermally.…”

The Fuzziness in Molecular, Supramolecular, and Systems Chemistry

“…Such PMLOs are fuzzy, and their fuzziness resides in their compositional and compartmental variety and variability. Dodero and her team made the tangible experience of supramolecular fuzziness by investigating the interaction between a Transcription Factor and double-stranded DNA [ 32 ]. After annealing a proper DNA sequence and synthesizing a photosensitive surrogate of the GCN4 Transcription Factor, Dodero and her colleagues furnish experimental evidence of the protein-DNA complexation fuzziness by using different techniques, such as NMR, electrophoretic mobility shift assay, and circular dichroism spectroscopy.…”