RESUMO
A detailed spectroscopic study (fluorescence, absorption, and lifetime) was conducted to gain insight into the nature of the binding interaction between fluorophore Hoechst33258 (H258) and jaundice marker Bilirubin (Br). The fluorescence emission of the H258 (Ex/Em = 340-502nm) showed a conc. dependent quenching in the presence of Br (1.25[Formula: see text]M to 10[Formula: see text]M). The Stern-Volmer constant demonstrated an upward curve depicting the occurrence of both static and dynamic quenching with an acquired value of K[Formula: see text] = 3.1x 10[Formula: see text] M[Formula: see text] and biomolecular quenching rate constant K[Formula: see text] = 8.6 x 10[Formula: see text] M[Formula: see text]S[Formula: see text]. The static quenching was evaluated using the sphere of action model and a sphere radius of 0.3nm indicated the presence of a static component in the quenching. The FRET analysis with overlap integral (J) = 1.4x10[Formula: see text] M[Formula: see text]cm[Formula: see text]nm[Formula: see text] and Foster Radius(R[Formula: see text]) = 26.82 Å with 59% efficiency suggested occurrence of dynamic quenching. Further studies with the time-resolved fluorescence also indicated the presence of dynamic quenching. The lifetime values of H258 reduced from 3.9ns to 0.5ns. Molecular docking studies further support both static and dynamic components in quenching. A non-covalent interaction of H258 with Br in the presence of HSA is predominantly characterized by H-bonding with residues Lys, Asn, Glu, Gln, and Br. The H258 and Br interaction was within the distance of 3.04 Å, which is in coherence with the sphere of action model (0.3nm) and Van-der-Waals along with hydrophobic interactions, which suggested both static and dynamic quenching. Thus, H258 can serve as an efficient fluorophore to monitor binding interactions and can be further exploited as a suitable probe for investigating conformational changes and detection of Br in subsequent studies.
RESUMO
Currently, we are at the threshold of the 'post-antibiotic era' due to the global emergence of antimicrobial resistance (AMR), and hence there is a dire need to discover new antibiotics. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a diverse class of natural products (NPs), some of which are under clinical trials for their antimicrobial potential. Thiopeptides are structurally one of the most complex classes of RiPPs due to numerous post-translational modifications (PTMs), with [4+2] cycloaddition being the core PTM and are active against several gram-positive pathogens. Genome mining coupled with experimental work can harness the unexplored 'cryptic' gene clusters while minimizing the rate of the rediscovery of known metabolites and expand the molecular diversity of NPs with medicinal potential. Employing the genome mining approach using a series of freely available bioinformatics tools, we have identified eight novel putative thiopeptide encoding biosynthetic gene clusters (BGCs) from different bacterial genomes, most of which belong to the class Actinobacteria. Our results provide confidence in the newly identified BGCs, to proceed with wet-bench experiments and discover novel thiopeptide(s).
