Significance of Secondary Structure Determination When Evaluating Rationally Designed Antimicrobial Peptides

In an effort to provide new treatments for the global crisis of bacterial resistance to current antibiotics, we have used a rational approach to design several new antimicrobial peptides. The present study focuses on 24-mer WLBU2 and its derivative D8. This amino acid sequence contains only R, W and V: RRWVRRVRRWVRRVVRVVRRWVRR. In D8, all of the valines are the D-enantiomer. WLBU2 and D8 have similar bactericidal activity, with low MIC values for both gram-negative (G(-)) and gram-positive (G(+)) bacteria. Secondary structure was determined using both circular dichroism (CD) and NMR. CD determined that WLBU2 is random in aqueous solution, but transitions to largely helical when interacting with a gram-negative (G(-)) membrane mimic, consisting of PE:PG:CL (7:2:1). With D8, the secondary structure could not be determined using CD since the D-enantiomers cause an inversion of the four typical structural motifs. Therefore, 13C labeled peptides were used to measure chemical shift differences of Cα-Cβ 2D with HSQC-TOCSY in solution. This confirmed the CD result that WLBU2 is random and determined that D8 is also random in solution. A solid state NMR method operating with magic angle spinning, DARR, was used to determine that WLBU2 is helical and D8 is random when embedded in G(-) mimics. While their secondary structures are very different in membranes, both WLBU2 and D8 kill similarly G(-) and G(+) bacteria, suggesting that secondary structure is not the prime consideration when evaluating bactericidal activity of newly designed AMPs. However, in terms of toxicity to red blood cells (RBCs) there was a dramatic difference, in that WLBU2 caused RBC hemolysis, while D8 did not. Therefore, secondary structure may play a role in eukaryotic cell toxicity. Other structural aspects of these peptides are now being investigated.