No abstract

Chionodracine (Cnd) is a 22-residue peptide of the piscidin family expressed in the gills of the Chionodraco hamatus as protection from bacterial infections. Here, we report the effects of synthetic Cnd on both Psychrobacter sp. TAD1 and Escherichia coli bacteria, as well as membrane models. We found that Cnd perforates the inner and outer membranes of Psychrobacter sp. TAD1, making discrete pores that cause the cellular content to leak out. Membrane disruption studies using intrinsic and extrinsic fluorescence spectroscopy revealed that Cnd behaves similarly to other piscidins, with comparable membrane partition coefficients. Membrane accessibility assays and structural studies using NMR in detergent micelles show that Cnd adopts a canonical topology of antimicrobial helical peptides, with the hydrophobic face toward the lipid environment and the hydrophilic face toward the bulk solvent. The analysis of Cnd free energy of binding to vesicles with different lipid contents indicates a preference for charged phospholipids and a more marked binding to native E. coli extracts. Taken with previous studies on piscidin-like peptides, we conclude that Cnd first adsorbs to the membrane, and then forms pores together with membrane fragmentation. Since Cnd has only marginal hemolytic activity, it constitutes a good template for developing new antimicrobial agents.

Water plays a central role in membrane protein folding and function. It not only catalyzes lipid membrane self-assembly but also affects the structural integrity and conformational dynamics of membrane proteins. Magic angle spinning (MAS) solid-state NMR (ssNMR) is the technique of choice for measuring water accessibility of membrane proteins, providing a measure for membrane protein topology and insertion within lipid bilayers. However, the sensitivity and resolution of membrane protein samples for MAS experiments are often dictated by hydration levels, which affect the structural dynamics of membrane proteins. Oriented-sample ssNMR (OS-ssNMR) is a complementary technique to determine both structure and topology of membrane proteins in liquid crystalline bilayers. Recent advancements in OS-ssNMR involve the use of oriented bicellar phases that have improved both sensitivity and resolution. Importantly, for bicelle formation and orientation, lipid bilayers must be well organized and hydrated, resulting in the protein's topology being similar to that found in native membranes. Under these conditions, the NMR resonances become relatively narrow, enabling a better separation of 1H–15N dipolar couplings and anisotropic 15N chemical shifts with separated local field (SLF) experiments. Here, we report a residue-specific water accessibility experiment for a small membrane protein, sarcolipin (SLN), embedded in oriented lipid bicelles as probed by new water-edited SLF (WE-SLF) experiments. We show that SLN's residues belonging to the juxtamembrane region are more exposed to the water–lipid interface than the corresponding membrane-embedded residues. The information that can be obtained from the WE-SLF experiments can be interpreted using a simple theoretical model based on spin-diffusion theory and offers a complete characterization of membrane proteins in realistic membrane bilayer systems.

To transmit signals across cellular compartments, many membrane-embedded enzymes undergo extensive conformational rearrangements. Monitoring these events in lipid bilayers by NMR at atomic resolution has been challenging due to the large size of these systems. It is further exacerbated for large mammalian proteins that are difficult to express and label with NMR-active isotopes. Here, we synthesized and engineered 13C ethyl groups on native cysteines to map the structural transitions of the sarcoplasmic reticulum Ca2+-ATPase, a 110 kDa transmembrane enzyme that transports Ca2+ into the sarcoplasmic reticulum. Using magic angle spinning NMR, we monitored the chemical shifts of the methylene and methyl groups of the derivatized cysteine residues along the major steps of the enzymatic cycle. The methylene chemical shifts are sensitive to the ATPase conformational changes induced upon nucleotide and Ca2+ ion binding and are ideal probes for active and inactive states of the enzyme. This new approach is extendable to large mammalian enzymes and signaling proteins with native or engineered cysteine residues in their amino acid sequence.

No abstract