Molecular Basis of Selectivity and Activity for the Antimicrobial Peptide Lynronne-1 Informs Rational Design of Peptide with Improved Activity.

Antibiotic resistance is a significant threat to human health, with natural products remaining the best source for new antimicrobial compounds. Antimicrobial peptides (AMPs) are natural products with great potential for clinical use as they are small, amenable to customization, and show broad-spectrum activities. Lynronne-1 is a promising AMP identified in the rumen microbiome that shows broad-spectrum activity against pathogens such as methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii. Here we investigated the structure of Lynronne-1 using solution NMR spectroscopy and identified a 13-residue amphipathic helix containing all six cationic residues. We used biophysical approaches to observe folding, membrane partitioning and membrane lysis selective to the presence of anionic lipids. We translated our understanding of Lynronne-1 structure to design peptides which varied in the size of their hydrophobic helical face. These peptides displayed the predicted continuum of membrane-lysis activities in vitro and in vivo, and yielded a new AMP with 4-fold improved activity against A. baumannii and 32-fold improved activity against S. aureus.

Nanoscale Surface Charge Visualization of Human Hair.

The surface charge and topography of human hair are visualized synchronously at the nanoscale using scanning ion conductance microscopy (SICM), a scanning nanopipette probe technique that uses local ion conductance currents to image the physicochemical properties of interfaces. By combining SICM data with finite element method (FEM) simulations that solve for ion transport at the nanopipette under bias, one is able to quantitatively correlate colocated surface charge and topography. The hair samples studied herein, from a 25-year-old Caucasian male with light hair (as an exemplar), reveal that untreated hair, in areas ca. 1 cm from the root, has a fairly uniform negative charge density of ca. -15 mC/cm-2 (in pH 6.8 aqueous solution), with some higher magnitude negative values localized near the boundaries between hair cuticles. Common chemical treatments result in varying degrees of charge heterogeneity. A bleach treatment produces some highly negatively charged localized regions (-80 to -100 mC/cm-2 at pH 6.8), due to hair damage, while a chemical conditioner treatment causes an overall increase in the homogeneity of the surface charge, together with a shift in the surface charge to positive values. Bleached surfaces are temporarily repaired to some extent through the use of a conditioner, as judged by the surface charge values. Finally, SICM is able to detect differences in the surface charge density of hair at different distances from the root (equivalent to hair age). Presently, the assessment of hair surface charge mainly relies on zeta-potential measurements which lack spatial resolution, among other drawbacks. In contrast, SICM enables quantitative surface charge mapping that should be beneficial in deepening understanding of the physicochemical properties of hair and lead to the rational development of new treatments and the assessment of their efficacy at the nanoscale. Given the widespread interest in the surface charge properties of interfaces, this work further demonstrates that SICM should generally become an important characterization tool for surface analytical chemists.

A C-terminal amphipathic helix is necessary for the in vivo tubule-shaping function of a plant reticulon.

Reticulons (RTNs) are a class of endoplasmic reticulum (ER) membrane proteins that are capable of maintaining high membrane curvature, thus helping shape the ER membrane into tubules. The mechanism of action of RTNs is hypothesized to be a combination of wedging, resulting from the transmembrane topology of their conserved reticulon homology domain, and scaffolding, arising from the ability of RTNs to form low-mobility homo-oligomers within the membrane. We studied the plant RTN isoform RTN13, which has previously been shown to locate to ER tubules and the edges of ER cisternae and to induce constrictions in ER tubules when overexpressed, and identified a region in the C terminus containing a putative amphipathic helix (APH). Here we show that deletion of this region or disruption of the hydrophobic face of the predicted helix abolishes the ability of RTN13 to induce constrictions of ER tubules in vivo. These mutants, however, still retain their ability to interact and form low-mobility oligomers in the ER membrane. Hence, our evidence indicates that the conserved APH is a key structural feature for RTN13 function in vivo, and we propose that RTN, like other membrane morphogens, rely on APHs for their function.

Curvature sensing amphipathic helix in the C-terminus of RTNLB13 is conserved in all endoplasmic reticulum shaping reticulons in Arabidopsis thaliana.

The reticulon family of integral membrane proteins are conserved across all eukaryotes and typically localize to the endoplasmic reticulum (ER), where they are involved in generating highly-curved tubules. We recently demonstrated that Reticulon-like protein B13 (RTNLB13) from Arabidopsis thaliana contains a curvature-responsive amphipathic helix (APH) important for the proteins' ability to induce curvature in the ER membrane, but incapable of generating curvature by itself. We suggested it acts as a feedback element, only folding/binding once a sufficient degree of curvature has been achieved, and stabilizes curvature without disrupting the bilayer. However, it remains unclear whether this is unique to RTNLB13 or is conserved across all reticulons-to date, experimental evidence has only been reported for two reticulons. Here we used biophysical methods to characterize a minimal library of putative APH peptides from across the 21 A. thaliana isoforms. We found that reticulons with the closest evolutionary relationship to RTNLB13 contain curvature-sensing APHs in the same location with sequence conservation. Our data reveal that a more distantly-related branch of reticulons developed a ~ 20-residue linker between the transmembrane domain and APH. This may facilitate functional flexibility as previous studies have linked these isoforms not only to ER remodeling but other cellular activities.

Revealing the mechanism of protein-lipid interactions for a putative membrane curvature sensor in plant endoplasmic reticulum.

Membrane curvature sensing via helical protein domains, such as those identified in Amphiphysin and ArfGAP1, have been linked to a diverse range of cellular processes. However, these regions can vary significantly between different protein families and thus remain challenging to identify from sequence alone. Greater insight into the protein-lipid interactions that drive this behavior could lead to production of therapeutics that specifically target highly curved membranes. Here we demonstrate the curvature-dependence of membrane binding for an amphipathic helix (APH) in a plant reticulon, namely RTNLB13 from A. thaliana. We utilize solution-state nuclear magnetic resonance spectroscopy to establish the exact location of the APH and map the residues involved in protein-membrane interactions at atomic resolution. We find that the hydrophobic residues making up the membrane binding site are conserved throughout all A. thaliana reticulons. Our results also provide mechanistic insight that leads us to propose that membrane binding by this APH may act as a feedback element, only forming when ER tubules reach a critical size and adding stabilization to these structures without disrupting the bilayer. A shallow hydrophobic binding interface appears to be a feature shared more broadly across helical curvature sensors and would automatically restrict the penetration depth of these structures into the membrane. We also suggest this APH is highly tuned to the composition of the membrane in which it resides, and that this property may be universal in curvature sensors thus rationalizing the variety of mechanisms reported for these functional elements.