Most antimicrobial peptides (AMPs) and their synthetic mimics (SMAMPs) are thought to act by permeabilizing cell membranes. For antimicrobial therapy, selectivity for pathogens over mammalian cells is a key requirement. Understanding membrane selectivity is thus essential for designing AMPs and SMAMPs to complement classical antibiotics in the future. This study focuses on membrane permeabilization induced by SMAMPs and their selectivity for membranes with different lipid compositions. We measure release and fluorescence lifetime of a self-quenching dye in lipid vesicles. Apart from the dose-response, we quantify the strength of individual leakage events, and, employing cumulative kinetics, categorize permeabilization behavior. We propose that differing selectivities in a series of SMAMPs arise from a combination of the effect of the antimicrobial agent and the susceptibility of the membrane (with a given lipid composition) for certain types of leakage behavior. The unselective and hemolytic SMAMP is found to act mainly by the asymmetry stress mechanism, mediated by hydrophobic insertion of SMAMPs into lipid layers. The more selective SMAMPs induced leakage events occurring stochastically over several hours. Lipid intrinsic properties might additionally amplify the efficiency of leakage events. Leakage behavior changes with both the design of the SMAMP and the lipid composition of the membrane. Understanding how leakage behavior contributes to the selectivity and activity of antimicrobial agents will aid the design and screening of antimicrobials. An understanding of the underlying processes facilitates the comparison of membrane permeabilization across in vitro and in vivo assays.
Abbreviations: CaM, calmodulin; CBD, CHP-binding region of sodium/proton exchanger 1 (residues 503 to 545); CHP, calcineurin B homologous protein; CV, column volumes; EFCaBPs, EF-hand Ca 2+ -binding proteins; FPH assay, fluorescent probe hydrophobicity assay; ITC, isothermal titration calorimetry; MBP, maltose-binding protein; MST, microscale thermophoresis; NHE1, sodium proton exchanger 1; PAGE, polyacrylamide gel electrophoresis. AbstractCalcineurin B homologous proteins (CHPs) belong to the EF-hand Ca 2+ -binding protein (EFCaBP) family. They have multiple important functions including the regulation of the Na + /H + exchanger 1 (NHE1). The human isoforms CHP1 and CHP2 share high sequence similarity, but have distinct expression profiles with CHP2 levels for instance increased in malignant cells. These CHPs bind Ca 2+ with high affinity.Biochemical data indicated that Ca 2+ can regulate their functions. Experimental evidence for Ca 2+ -modulated structural changes was lacking. With a newly established fluorescent probe hydrophobicity (FPH) assay, we detected Ca 2+ -induced conformational changes in both CHPs. These changes are in line with an opening of their hydrophobic pocket that binds the CHP-binding region (CBD) of NHE1. Whereas the pocket is closed in the absence of Ca 2+ in CHP2, it is still accessible for the dye in CHP1. Both CHPs interacted with CBD in the presence and absence of Ca 2+ .Isothermal titration calorimetry (ITC) analysis revealed high binding affinity for both CHPs to CBD with equilibrium dissociation constants (K D s) in the nanomolar range.The K D for CHP1:CBD was not affected by Ca 2+ , whereas Ca 2+ -depletion increased the K D 7-fold for CHP2:CBD showing a decreased affinity. The data indicate an isoform specific regulatory interaction of CHP1 and CHP2 with NHE1. K E Y W O R D Scalcineurin B homologous proteins, Ca 2+ signaling, EF-hand Ca 2+ -binding proteins, NHE1, proteinprotein interaction 3254 | LIANG et AL.
The interactions of exenatide, a Trp-containing peptide used as a drug to treat diabetes, with liposomes were studied by isothermal titration calorimetry (ITC), tryptophan (Trp) fluorescence, and microscale thermophoresis measurements. The results are not only important for better understanding the release of this specific drug from vesicular phospholipid gel formulations but describe a general scenario as described before for various systems. This study introduces a model to fit these data on the basis of primary and secondary peptide-lipid interactions. Finally, resolving apparent inconsistencies between different methods aids the design and critical interpretation of binding experiments in general. Our results show that the net cationic exenatide adsorbs electrostatically to liposomes containing anionic diacyl phosphatidylglycerol lipids (PG); however, the ITC data could not properly be fitted by any established model. The combination of electrostatic adsorption of exenatide to the membrane surface and its self-association (K d ¼ 46 mM) suggested the possibility of secondary binding of peptide to the first, primarily (i.e., lipid-) bound peptide layer. A global fit of the ITC data validated this model and suggested one peptide to bind primarily per five PG molecules with a K d z 0.2 mM for PC/PG 1:1 and 0.6 mM for PC/PG 7:3 liposomes. Secondary binding shows a weaker affinity and a less exothermic or even endothermic enthalpy change. Depending on the concentration of liposomes, secondary binding may also lead to liposomal aggregation as detected by dynamic light-scattering measurements. ITC quantifies primary and secondary binding separately, whereas microscale thermophoresis and Trp fluorescence represent a summary or average of both effects, possibly with the fluorescence data showing somewhat greater weighting of primary binding. Systems with secondary peptide-peptide association within the membrane are mathematically analogous to the adsorption discussed here.
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