RESUMO
Antisense peptide nucleic acids (PNAs) inhibit bacterial growth in several infection models. Since PNAs are not spontaneously taken up by bacteria, they are often conjugated to carriers such as cell-penetrating peptides (CPPs) in order to improve translocation. Hydrophobic counterions such as pyrenebutyrate (PyB) have been shown to facilitate translocation of peptides over natural and artificial membranes. In this study, the capability of PyB to support translocation of CPP-coupled antisense PNAs into bacteria was investigated in Streptococcus pyogenes and Streptococcus pneumoniae. PyB enhanced the antimicrobial activity of CPP-conjugated antisense PNAs in S. pyogenes. The most significant effect of PyB was observed in combination with K8-conjugated anti-gyrA PNAs. In contrast, no significant effect of PyB on the antimicrobial activity of CPP-conjugated PNAs in S. pneumoniae was detected. Uptake of K8-FITC into S. pyogenes, Escherichia coli, and Klebsiella pneumoniae could be improved by pre-incubation with PyB, indicating that PyB supports the antimicrobial effect of CPP-antisense PNAs in S. pyogenes by facilitating the translocation of peptides across the bacterial membrane.
RESUMO
Protein-lipid interactions are important for folding and membrane insertion of integral membrane proteins that are composed either of α-helical or of ß-barrel structure in their transmembrane domains. While α-helical transmembrane proteins fold co-translationally while they are synthesized by a ribosome, ß-barrel transmembrane proteins (ß-TMPs) fold and insert posttranslationally-in bacteria after translocation across the cytoplasmic membrane, in cell organelles of eukaryotes after import across the outer membrane of the organelle. ß-TMPs can be unfolded in aqueous solutions of chaotropic denaturants like urea and spontaneously refold upon denaturant dilution in the presence of preformed lipid bilayers. This facilitates studies on lipid interactions during folding into lipid bilayers. For several ß-TMPs, the kinetics of folding has been reported as strongly dependent on protein-lipid interactions. The kinetics of adsorption/insertion and folding of ß-TMPs can be monitored by fluorescence spectroscopy. These fluorescence methods are even more powerful when combined with site-directed mutagenesis for the preparation of mutants of a ß-TMP that are site-specifically labeled with a fluorophore or a fluorophore and fluorescence quencher or fluorescence resonance energy acceptor. Single tryptophan or single cysteine mutants of the ß-TMP allow for the investigation of local protein-lipid interactions, at specific regions within the protein. To examine the structure formation of ß-TMPs in a lipid environment, fluorescence spectroscopy has been used for double mutants of ß-TMPs that contain a fluorescent tryptophan and a spin-label, covalently attached to a cysteine as a fluorescence quencher. The sites of mutation are selected so that the tryptophan is in close proximity to the quencher at the cysteine only when the ß-TMP is folded. In a folding experiment, the evolution of fluorescence quenching as a function of time at specific sites within the protein can provide important information on the folding mechanism of the ß-TMP. Here, we report protocols to examine membrane protein folding for two ß-TMPs in a lipid environment, the outer membrane protein A from Escherichia coli, OmpA, and the voltage-dependent anion-selective channel, human isoform 1, hVDAC1, from mitochondria.
