RESUMO
Antimicrobial activity of many AMPs can be improved by lysine-to-arginine substitution due to a more favourable interaction of arginine guanidinium moiety with bacterial membranes. In a previous work, the structural and functional characterization of an amphipathic antimicrobial peptide named RiLK1, including lysine and arginine as the positively charged amino acids in its sequence, was reported. Specifically, RiLK1 retained its ß-sheet structure under a wide range of environmental conditions (temperature, pH, and ionic strength), and exhibited bactericidal activity against Gram-positive and Gram-negative bacteria and fungal pathogens with no evidence of toxicity on mammalian cells. To further elucidate the influence of a lysine-to-arginine replacement on RiLK1 conformational properties, antimicrobial activity and peptide-liposome interaction, a new RiLK1-derivative, named RiLK3, in which the lysine is replaced with an arginine residue, was projected and characterised in comparison with its parental compound. The results evidenced that lysine-to-arginine mutation not only did not assure an improvement in the antimicrobial potency of RiLK1 in terms of bactericidal, virucidal and fungicidal activities, but rather it was completely abolished against the hepatitis A virus. Therefore, RiLK1 exhibited a wide range of antimicrobial activity like other cationic peptides, although the exact mechanisms of action are not completely understood. Moreover, tryptophan fluorescence measurements confirmed that RiLK3 bound to negatively charged lipid vesicles with an affinity lower than that of RiLK1, although no substantial differences from the structural and self-assembled point of view were evidenced. Therefore, our findings imply that antimicrobial efficacy and selectivity are affected by several complex and interrelated factors related to substitution of lysine with arginine, such as their relative proportion and position. In this context, this study could provide a better rationalisation for the optimization of antimicrobial peptide sequences, paving the way for the development of novel AMPs with broad applications.
RESUMO
Generation of the 3' overhang is a critical step during homologous recombination (HR) and replication fork rescue processes. This event is usually performed by a series of DNA nucleases and/or helicases. The nuclease NurA and the ATPase HerA, together with the highly conserved MRE11/RAD50 proteins, play an important role in generating 3' single-stranded DNA during archaeal HR. Little is known, however, about HerA-NurA function and activation of this fundamental and complicated DNA repair process. Herein, we analyze the functional relationship among NurA, HerA and the single-strand binding protein SSB from Saccharolubus solfataricus. We demonstrate that SSB clearly inhibits NurA endonuclease activity and its exonuclease activities also when in combination with HerA. Moreover, we show that SSB binding to DNA is greatly stimulated by the presence of either NurA or NurA/HerA. In addition, if on the one hand NurA binding is not influenced, on the other hand, HerA binding is reduced when SSB is present in the reaction. In accordance with what has been observed, we have shown that HerA helicase activity is not stimulated by SSB. These data suggest that, in archaea, the DNA end resection process is governed by the strictly combined action of NurA, HerA and SSB.
