Abstract:
β-lactam antibiotics are the most commonly used antibiotics due to their high effectiveness, low cost, ease of delivery and minimal side effects. Bacteria have acquired resistance to these antibiotics. One of the mechanisms of acquired drug resistance is the bacterial production of beta-lactamases, which break down these antibiotics. Currently used beta-lactamase inhibitors are not effective at targeting the 700 types and new mutants of β-lactamases. β-lactamase inhibitor design is therefore an important research field. β-lactamase inhibitory protein (BLIP) of Streptomyces clavuligerus is a potent inhibitor of several β-lactamases. The research presented in this thesis aims to guide the design of new peptide inhibitors based on BLIP structures. Computational studies are performed to elucidate the mechanism of TEM-1 β-lactamase inhibiton by BLIP. To this end, molecular dynamics simulations of TEM-1 BLIP and TEM-1 peptide complexes were carried out. Asp49 residue on BLIP and the BLIP based peptide were mutated to determine the contribution of this residue to binding. Binding free energy of the TEM-1 BLIP and TEM-1 mutant BLIP (D49A) complexes were estimated by the molecular mechanics Poisson Boltzmann Surface Area method (MM-PBSA). The binding free energy of TEM-1 BLIP complex was lower than that of the TEM-1 mutant BLIP complex, which indicates the contribution of the Asp49 residue of BLIP to tight binding. The experimental studies aimed to test the in-vivo BLIP – RTEM-1 β-lactamase binding and inhibition. The interaction of the two proteins was already verified using in-vitro studies however there was no report on their interaction under physiological conditions within the cells. For this purpose BLIP gene from Streptomyces clavuligerus was inserted into the pET20b(+) plasmid vector downstream from the pelB signal sequence and the recombinant protein was expressed in E. coli BL21(DE3). This signal sequence was required to direct BLIP to the periplasmic space where the RTEM-1 β-lactamase was located. The β-lactamase was expressed constitutively from the same plasmid. Upon induction with IPTG, cell growth was slightly retarded. This may be an evidence of the β-lactamase inhibition by BLIP and may provide preliminary results for further in-vivo experiments about BLIP β-lactamase binding.
