The interaction between host-defense antimicrobial peptides (AMPs) and the bacterial lipopolysaccharide (LPS) governs both the susceptibility of the bacteria to the peptide and the ability of the peptide to inhibit LPS activation of immune cells. Both functions depend on the biophysical properties of the peptides. However, the sequence and structural diversity of AMPs makes it difficult to determine common denominators required for antimicrobial and LPS neutralizing activities. Toward this end, we synthesized and investigated a series of nine 12-amino acid peptides and their fatty acid-conjugated analogues composed of both D- and L-isomers of Leu and Lys at various ratios. The positions of the D-amino acids were preserved. These peptides differ in their net positive charge and hydrophobicity. However, their overall structure in the membrane is similar, as determined by Fourier transform infrared spectroscopy. The peptides and their analogues were functionally tested for their antibacterial and hemolytic activity, their ability to permeate LPS vesicles, their ability to neutralize LPS activation of macrophages, and their effect on LPS morphology, determined by negative staining electron microscopy. The data revealed that increasing the ratio between hydrophobicity and the net positive charge increases both antimicrobial and LPS neutralization activities, but with different modes of contributions. Whereas antimicrobial activity increases linearly with the increase in the peptides' hydrophobicity, peptides with different hydrophobicities are endowed with similar LPS neutralizing activities. Besides adding important information regarding AMP parameters involved in antimicrobial and anti-LPS activities, this study suggests the use of such diastereomers as potential templates for the development of simple molecules that conduct both types of functions.

Membrane fusion between the human immunodeficiency virus (HIV) and the target cell plasma membrane is correlated with conformational changes in the HIV gp41 glycoprotein, which include an early exposed conformation (prehairpin) and a late low energy six helix bundle (SHB) conformation also termed hairpin. Peptides resembling regions from the exposed prehairpin have been previously studied for their interaction with membranes. Here we report on the expression, purification, SHB stability, and membrane interaction of the full-length ectodomain of the HIV gp41 and its two deletion mutants, all in their SHB-folded state. The interaction of the proteins with zwitterionic and negatively charged membranes was examined by using various biophysical methods including circular dichroism spectroscopy, differential scanning calorimetry, lipid mixing of large unilamellar vesicles, and atomic force microscopy (AFM). All experiments were done in an acidic environment in which the protein remains in its soluble trimeric state. The data reveal that all three proteins fold into a stable coiled-coil core in aqueous solution and retain a stable helical fold with reduced coiled-coil characteristics in a zwitterionic and negatively charged membrane mimetic environment. Furthermore, in contrast with the extended exposed N-terminal domain, the folded gp41 ectodomain does not induce lipid mixing of zwitterionic membranes. However, it disrupts and induces lipid mixing of negatively charged phospholipid membranes (approximately 100-fold more effective than fusion peptide alone), which are known to be expressed more in HIV-1-infected T cells or macrophages. The results support the emerging model in which one of the roles of gp41 folding into the SHB conformation is to slow down membrane disruption effects induced by early exposed gp41. However, it can further affect membrane morphology once exposed to negatively charged membranes during late stages.

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