Probing Molecular Interactions between Surface-Immobilized Antimicrobial Peptides and Lipopolysaccharides In Situ.

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. Recently, a label-free immobilized antimicrobial peptide (AMP) surface plasmon resonance platform was developed to successfully distinguish LPS from multiple bacterial strains. Among the tested AMPs, SMAP29 exhibited excellent affinity with LPS and has two independent LPS-binding sites located at two termini of the peptide. In this study, sum frequency generation vibrational spectroscopy was applied to investigate molecular interactions between three LPS samples and surface-immobilized SMAP29 via the N-terminus, the C-terminus, and a middle site at the solid/liquid interface in situ in real-time, supplemented by circular dichroism spectroscopy. It was found that the conformations and orientations of surface-immobilized SMAP29 via different sites are different when interacting with the same LPS, with different interaction kinetics. The same SMAP29 sample also has different structures and interaction kinetics while interacting with different LPS samples with different charge densities and hydrophobicities. The observed results on molecular interactions between surface-immobilized peptides and LPS can well interpret the different adsorption amounts of various LPSs on different surface-immobilized peptides.

Differential presentation of a single antimicrobial peptide is sufficient to identify LPS from distinct bacterial samples.

Rapid detection and identification of bacteria is important for human health, biodefense, and food safety. Small arrays of different antimicrobial peptides (AMPs) enable the identification of lipopolysaccharide (LPS) samples from a variety of bacterial species and strains. A model system for examining how peptide presentation affects LPS detection is the sheep myeloid antimicrobial peptide (SMAP-29), which contains a helix-turn-helix motif. Varying the cysteine attachment site on SMAP-29 controls the three-dimensional presentation of the peptide on the surface, altering the ability of the peptide to discriminate between LPS samples. A small array of only SMAP-29 variants-and no other peptides-is capable of discriminating among LPS samples from multiple bacterial species, as well as between different strains within the same species, with high accuracy.

Trimerization of the HIV Transmembrane Domain in Lipid Bilayers Modulates Broadly Neutralizing Antibody Binding.

The membrane-proximal external region (MPER) of HIV gp41 is an established target of antibodies that neutralize a broad range of HIV isolates. To evaluate the role of the transmembrane (TM) domain, synthetic MPER-derived peptides were incorporated into lipid nanoparticles using natural and designed TM domains, and antibody affinity was measured using immobilized and solution-based techniques. Peptides incorporating the native HIV TM domain exhibit significantly stronger interactions with neutralizing antibodies than peptides with a monomeric TM domain. Furthermore, a peptide with a trimeric, three-helix bundle TM domain recapitulates the binding profile of the native sequence. These studies suggest that neutralizing antibodies can bind the MPER when the TM domain is a three-helix bundle and this presentation could influence the binding of neutralizing antibodies to the virus. Lipid-bilayer presentation of viral antigens in Nanodiscs is a new platform for evaluating neutralizing antibodies.

Tubulin engineering by semi-synthesis reveals that polyglutamylation directs detyrosination.

Microtubules, a critical component of the cytoskeleton, carry post-translational modifications (PTMs) that are important for the regulation of key cellular processes. Long-lived microtubules, in neurons particularly, exhibit both detyrosination of α-tubulin and polyglutamylation. Dysregulation of these PTMs can result in developmental defects and neurodegeneration. Owing to a lack of tools to study the regulation and function of these PTMs, the mechanisms that govern such PTM patterns are not well understood. Here we produce fully functional tubulin carrying precisely defined PTMs within its C-terminal tail. We ligate synthetic α-tubulin tails-which are site-specifically glutamylated-to recombinant human tubulin heterodimers by applying a sortase- and intein-mediated tandem transamidation strategy. Using microtubules reconstituted with these designer tubulins, we find that α-tubulin polyglutamylation promotes its detyrosination by enhancing the activity of the tubulin tyrosine carboxypeptidase vasohibin/small vasohibin-binding protein in a manner dependent on the length of polyglutamyl chains. We also find that modulating polyglutamylation levels in cells results in corresponding changes in detyrosination, corroborating the link between the detyrosination cycle to polyglutamylation.