Santoro, Federica (2023) Conformational and interaction studies through solution NMR and other biophysical techniques. [Tesi di dottorato]

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Abstract

Biophysics is an interdisciplinary field that combines the principles of physics and biology to study the physical and chemical properties of biological systems. During my PhD, I applied biophysical techniques, mainly nuclear magnetic resonance (NMR) and circular dichroism (CD), but also X-ray crystallography, aimed at investigating different systems of pharmacological and chemical interest. They can be grouped in: 1) conformational analysis of bioactive peptides; 2) interaction analysis between small-molecules and DNA; 3) molecular interactions in a micellar reaction environment; 4) bacterial protein–protein interactions. These studies are briefly described below.I have carried on a conformational analysis of novel β-hairpin peptides derived from the transcription factor protein, ARC repressor. Transcription factors (TFs) have a remarkable role in the homeostasis of the organisms and there is a growing interest in how they recognize and interact with specific DNA sequences. In particular, ARC repressors, form dimers that insert antiparallel β-sheets into the major groove of DNA. The 3D conformational arrangement of the investigated peptides was preliminarily verified through circular dichroism (CD). The peptides, found to be compatible with a β-hairpin folding (% of β-sheet structure > 30%), were analyzed using NMR, confirming a clear tendency of all peptides to adopt the β- hairpin structure. Finally, NMR-based structure calculation was also performed on the peptide which showed the most interesting binding and selectivity properties, and the simulated annealing gave a quite well-defined β-hairpin structure with a type I’ β-turn on its tip. In conclusion, we designed a novel β-hairpin peptide derived from the ARC repressor that selectively interacts with the major groove of B-DNA. I explored the solution conformation of novel cyclic peptides bearing the 1,4- and 1-5-disubstituted [1,2,3]-triazolyl moieties which replaced the disulfide bond of a known potent peptide antagonist of CXCR4. CXCR4 is a G-protein coupled receptor (GPCR), which modulates many physiological functions by interacting with its own ligand C-X-C motif chemokine 12 (CXCL12). The CXCR4/CXCL12 pair has emerged as key players in promoting the tumor growth, invasion, angiogenesis and metastasis in more than 30 different human cancers. Several strategies to hamper the interaction between CXCR4 and CXCL12 have been investigated, leading to the development of potent CXCR4 antagonists including cyclopeptides. The investigation of these novel cyclic peptides led to the identification of an agonist and an antagonist showing high affinity and selectivity against CXCR4. Their different efficacy was rationalized by NMR and computational studies that showed slight differences in the binding mode of the two peptides, which might account for the distinct ability in modulating CXCR4. I explored the secondary structure of three glucosylated peptides derived from proteins that have been selected by a bioinformatic approach for their conformational (OMGp and RTN4R) or sequence (FAN) homology with CSF114(Glc), a synthetic antigen which is specifically recognized by antibodies (Abs) in Multiple Sclerosis (MS) patients' sera. CD measurements indicated that the glucopeptides derived from OMGp and RTN4R featured a high percentage of β-strand content, as observed in the lead compound CSF114(Glc), while the peptide derived from FAN is mainly in a random coil conformation. Notably, β-strand content paralleled glucopeptide ability in Abs recognition. Notably, the progression of MS is also linked to exogenous infectious agents expressing antigenic molecules. In this context, MS antibodies recognize a cell-surface adhesin protein of non-typeable Haemophilus influenzae (NTHi) termed HMW1. Interestingly, peptides derived from OMGp and RTN4R, as well as CSF114(Glc), were able to cross-react with antibodies recognizing HMW1, thus confirming the presence of a shared epitope. The structural correspondence between an exogenous protein and a physiological one, is the basis of the hypothesis that molecular mimicry triggers the breakdown of self-tolerance in MS. Moreover, my results suggest that the resemblance among the MS-specific epitopes has a significant conformational component.Alongside, I performed conformational analysis of synthetic short-chain peptide analogues of Relaxin. The peptide hormone Relaxin (RLX) holds great promise as a cardiovascular and anti-fibrotic agent but is limited by the pharmacokinetic issues. Six low molecular weight peptides were designed with the objective to obtain RLX analogues with improved pharmacokinetic features. Their secondary structure propensity was explored by CD spectroscopy. CD spectra of the peptides showed a tendency to assume α-helical secondary structure, typical of the hormone bioactive conformation. Despite the favourable premises, none of the tested peptides revealed a substantial affinity to RLX-receptor, RXFP1, nor displayed any RLX-like biological effects. Albeit negative, these results offer additional information about the structural requirements that new peptide therapeutics shall possess to effectively behave as RXFP1 agonists and RLX analogues. I have investigated the ability of novel designed compounds to interact with noncanonical DNAs, such as G-quadruplexes (G4s) and i-motifs (iMs), whose occurrence in gene promoters, replication origins, and telomeres highlights the breadth of biological processes that they might regulate. In particular, STD NMR experiments were performed. STD NMR spectra demonstrated a selective interaction between some of the developed compounds and noncanonical DNAs. Specifically, the presence of both aromatic and aliphatic signals in the STD spectra suggests that the aromatic moieties of some compounds could interact with the G4 and iM through π-π stacking with the guanines of G-tetrads and the adenines of loops, respectively, while the side chains could interact with the backbone of the DNA molecules through electrostatic interactions. Our investigation led to the identification of novel compounds that might be used as tools to shed light on the mechanisms underlying the controversial biological roles of G4 and iM structures as well as on their intricated relationships. NMR can provide information about the structure, dynamics, and interactions of molecules in a reaction environment. Particularly, I have performed NMR experiments to study the reaction environment at the atomic level of a photo-micellar catalyzed synthesis of amides from isocyanides. I studied the localization of the photocatalyst [Ir(ppy)2bpy]PF6 relative to the surface and the interior of two micellar systems SDS or CTAC which turned out to be the most and the least efficient reaction systems, respectively. The study was carried out determining the NOE contacts between photocatalyst and the micelles and using specific paramagnetic probes, 16- doxylstearic acid (16-doxyl) and Mn2+, for the position determination. NMR analysis demonstrated that the catalyst is on average positioned on the micelle surface and can flip from the outer to inner part of it in the case of SDS while it is deeply inserted in CTAC micelles. The obtained experimental data allow a rational approach for selecting the best reaction conditions suggesting that, for an optimal catalytic efficiency, the photocatalyst must be positioned on the micelles’ surface and almost free to move inside and outside the micelles. Finally, X-ray crystallography can be used to study the structure of proteins, as well as their interactions with other molecules. I applied X-ray crystallography to investigate the binding mode of antitoxin-mimicking peptides derived from the protein Phd and the bacterial toxin Doc. One of the major challenges of studying toxin-antitoxin modules is to obtain good amounts of protein. In fact, in bacterial expression systems, toxin expression severely inhibits normal growth, resulting in production of only trace amounts of the toxin of interest. To overcome this issue, I applied a protocol of co-expression of Doc with Phd peptides that allowed us to obtain sufficient amounts of complex to be used for X-ray crystallography. The structure of the Doc-Phd complex helped us to confirm the molecular details of their interaction. The next step is to obtain the structure of Doc with a higher affinity peptide, in order to proceed with the comparison of the two binding modes aimed at designing novel antitoxin compounds useful as antibacterial weapons.

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