AcrB homológ membrán transzporterek vizsgálata = Investigation of AcrB homologue membrane transporters

Különböző AcrB homológ fehérjéket vizsgáltunk. Kettő a Brucella melitensis baktériumban kódolt (BMEI 1645, BMEI 0895) egy-egy pedig Haemophilus influenzae-ban (HI 0895). Acinetobacter baumannii-ban (ACIAD0783), Pseudomonas aeruginosa-ban (PA0158), Pectobacterium carotovorum-ban (ECA1169), Helicobacter pylori-ban (HP0607), Klebsiella pneumoniae-ban (KPN00443) valamint Bacillus cereus törzsekből: Bacillus cereus ATCC 10987 (BCE 0788), és Bacillus cereus ATCC 14579 (BC0714). Sikeres klónozást a következő gének esetén értünk el: BMEI 1645, BMEI 0895, HI 0895, BCE 0788, BC0714. A felsorolt gének közül a BMEI 1645-ös fehérje over-expresszálódott egyedül Escherichia coliban. Ezt a fehérjét szolubilizáltuk, tisztítottuk affinitás oszlopon, majd gélszűréssel. A fehérje két fő populációt mutatott a gélszűrés eredményként, melyeket jellemeznünk szükséges a fehérje kristályosítása előtt. Az AcrB fehérjét, mint kontrollt, sikeresen klónoztuk, expresszáltuk Escherichia coliban, nagy mennyiségben tisztítottuk és kristályosítottuk. A kristályosítás során ß-peptid foldamerek hatását vizsgáltuk és megállapítottuk, hogy a kristályosítás során segédanyagként használt foldamer megkönnyíteni, lerövidíteni látszik kristályosításhoz szükséges időt. | Different AcrB homologue transporters were examined. Two of them are coded in Brucella melitensis (BMEI 1645, BMEI 0895), one each is coded in Haemophilus influenzae (HI 0895). Acinetobacter baumannii (ACIAD0783), Pseudomonas aeruginosa (PA0158), Pectobacterium carotovorum (ECA1169), Helicobacter pylori (HP0607), Klebsiella pneumoniae (KPN00443) and two of them from different Bacillus cereus strains Bacillus cereus ATCC 10987 (BCE 0788) and Bacillus cereus ATCC 14579 (BC0714). The following genes were successfully cloned: BMEI 1645, BMEI 0895, HI 0895, BCE 0788, BC0714. BMEI 1645 protein was overexpressed in Escherichia coli. The protein was solubilized and purified by affinity chromatography and size exclusion chromatography. Two major populations of protein were observed after size exclusion chromatography and those need to be characterized before protein crystallization. AcrB protein as a control was successfully cloned, expressed in Escherichia coli, purified in large scale and crystallized. The effect of a ß-peptid foldamer was investigated and we determined that the ß-peptid foldamer as a crystallization adjuvant might help to decrease the time necessary for crystal formation

Recent insight into strategies for the design of antimicrobial peptides (AMPs)

With the increasing development of antibiotic resistance among key bacterial pathogens, there is an urgent need to discover novel classes of antibiotics. Although antimicrobial peptides (AMP) with their specific mode of action are considered major candidates for next-generation antibiotics, several challenges limit the use of these peptides for therapeutic applications. In a large body of research, the focus is given to different approaches to the chemical modification of AMPs and how these modifications may improve the stability, antibiotic activity, proteolytic activity and prevent the cytotoxicity and side effects of AMPs. On the other hand, another group of research investigates the delivery of AMPs via nanocarrier systems as strategies used to enhance stability, control the release of peptides and reduce adverse peptide-related side effects, as well as improve their anti-microbial activities. In the present article, we surveyed most recently published researches that provide us with good knowledge on structural features, mechanism of action, therapeutic aim, advantages and limitations, chemical modification approaches and carrying strategies of AMPs. Finally, according to Quality by Design, the most important potential effective factor and potential risk were mentioned in the development of AMP delivery systems

Structural Adaptation of the Single-Stranded DNA-Binding Protein C-Terminal to DNA Metabolizing Partners Guides Inhibitor Design

Single-stranded DNA-binding protein (SSB) is a bacterial interaction hub and an appealing target for antimicrobial therapy. Understanding the structural adaptation of the disordered SSB C-terminus (SSB-Ct) to DNA metabolizing enzymes (e.g., ExoI and RecO) is essential for designing high-affinity SSB mimetic inhibitors. Molecular dynamics simulations revealed the transient interactions of SSB-Ct with two hot spots on ExoI and RecO. The residual flexibility of the peptide–protein complexes allows adaptive molecular recognition. Scanning with non-canonical amino acids revealed that modifications at both termini of SSB-Ct could increase the affinity, supporting the two-hot-spot binding model. Combining unnatural amino acid substitutions on both segments of the peptide resulted in enthalpy-enhanced affinity, accompanied by enthalpy–entropy compensation, as determined by isothermal calorimetry. NMR data and molecular modeling confirmed the reduced flexibility of the improved affinity complexes. Our results highlight that the SSB-Ct mimetics bind to the DNA metabolizing targets through the hot spots, interacting with both of segments of the ligands