Abstract
We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.
Highlights
Antimicrobial peptides (AMPs) have the potential to act against multiple pathogenic targets
We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties
When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis
Summary
Peptides—Peptides comprising L-amino acids (Table 1) were purchased from either EZBiolab (Carmel, IN) or Pepceuticals Ltd. (Nottingham, UK) as desalted grade. E. coli NCTC 9001, P. aeruginosa PAO1, or competent E. coli TOP10 were grown without shaking in 50 ml of Mueller-Hinton broth at 37 °C. In each of columns 2–11, 50 l of Mueller-Hinton broth was added under sterile conditions. 50 l of 256 g/ml stock peptide solutions prepared in distilled water were added, and the broth from the second row was pipetted into the first row and thoroughly mixed before being deposited again in the second row. This process was repeated throughout the tray, providing a 2-fold dilution of peptide with each row. The MIC50 quoted for each peptide (Table 2) is an average value from at least two independent repeats
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