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Membrane proteins are vital for cell function and thus represent important drug targets. Solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy offers a unique access to probe the structure and dynamics of such proteins in biological membranes of increasing complexity. Here, we present modern solid-state NMR spectroscopy as a tool to study structure and dynamics of proteins in natural lipid membranes and at atomic scale. Such spectroscopic studies profit from the use of high-sensitivity ssNMR methods, i.e., proton-( 1 H)-detected ssNMR and DNP (Dynamic Nuclear Polarization) supported ssNMR. Using bacterial outer membrane beta-barrel protein BamA and the ion channel KcsA, we present methods to prepare isotope-labeled membrane proteins and to derive structural and motional information by ssNMR.

NK-lysins, a type of broad-spectrum antimicrobial peptide (AMP), act as an essential effector of innate defense against microbial attack in higher vertebrates and so in fish. The present study delineates the structural and functional characterization of NK-lysin from yellow catfish (Pelteobagrus fulvidrac) (Pelteobagrus fulvidraco). PfNK-lysin encodes a 153-residue peptide, which displays the hallmark features of other known NK-lysins with the ordered array of six well-conserved cysteine residues and five-exon/four-intron structure. It was found to be ubiquitous in tissues, being detected most abundantly in gill and head kidney. In vivo exposure to stimuli (LPS, PolyI:C, and Edwardsiella ictaluri) induced PfNK-lysin expression in head kidney and spleen. Synthetic PfNK-lysin-derived peptide exhibited in vitro bactericidal potency against both Gram-positive and Gram-negative bacteria, with the highest inhibitory effect on pathogen Edwardsiella ictaluri. Fluorescence microscopy and scanning electron microscopy further confirmed its capacity to cause damage to the bacterial plasma membrane. Taken together, these data suggest that PfNK-lysin might participate in antimicrobial defense of yellow catfish by membrane-disruptive action., (Copyright © 2019. Published by Elsevier Ltd.)

Phospholipid scramblases catalyze the rapid trans-bilayer movement of lipids down their concentration gradients. This process is essential for numerous cellular signaling functions including cell fusion, blood coagulation, and apoptosis. The importance of scramblases is highlighted by the number of human diseases caused by mutations in these proteins. Because of their indispensable function, it is essential to understand and characterize the molecular function of phospholipid scramblases. Powerful tools to measure lipid transport in cells are available. However, these approaches provide limited mechanistic insights into the molecular bases of scrambling. Here we describe in detail an in vitro phospholipid scramblase assay and the accompanying analysis which allows for determination of the macroscopic rate constants associated with phospholipid scrambling. Notably, members of the TMEM16 family of scramblases also function as nonselective ion channels. To better understand the physiological relevance of this channel function as well as its relationship to the scrambling activity of the TMEM16s we also describe in detail an in vitro flux assay to measure nonselective channel activity. Together, these two assays can be used to investigate the dual activities of the TMEM16 scramblases/nonselective channels.

Membrane fusion mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-family proteins is an essential process for intracellular membrane trafficking in all eukaryotic cells, which delivers proteins and lipids to their appropriate subcellular membrane compartments such as organelles and plasma membrane. The molecular basis of SNARE-mediated membrane fusion has been revealed by studying fusion of reconstituted proteoliposomes bearing purified SNARE-family proteins and chemically defined lipid species. This chapter describes the detailed experimental protocols for (1) purification of recombinant SNARE-family and SM (Sec1/Munc18-family) proteins in the yeast Saccharomyces cerevisiae; (2) preparation of reconstituted proteoliposomes bearing purified yeast SNARE proteins; and (3) developing an assay to monitor lipid mixing between reconstituted SNARE-bearing proteoliposomes. Lipid mixing assays for reconstituted SNARE-bearing proteoliposomes are useful for evaluating the intrinsic capacity of SNARE-family proteins to directly catalyze membrane fusion and to determine the specificity of membrane fusion.

Tuberculosis (TB) is one of the most important causes of mortality and morbidity due to infectious diseases. BCG, the vaccine in use, is not fully protective against TB. In a previous study, we have shown that proteoliposomes (outer membrane extracts), obtained from BCG (PLBCG) were able to induce humoral immune responses against Mycobacterium tuberculosis (Mtb) antigens. With the objective to evaluate the protective capability of PLBCG alone or as a booster with BCG, a murine model of progressive pulmonary TB was used. Animals immunized with PLBCG adjuvanted with alum (PLBCG-Al) showed similar protection to that conferred by BCG. The group immunized with PLBCG-Al as a booster to BCG gave superior protection than BCG as evidenced by a reduction of bacterial load in lungs 2 months after infection with Mtb. Animals immunized with BCG, PLBCG-Al and this formulation as a booster of BCG, showed a significant decrease of tissue damage (percentage of pneumonic area/lung) compared with non-immunized animals. These results demonstrate that immunization with PLBCG-Al alone or as a booster to BCG induce appropriate protection against challenge with Mtb in mice and support the future evaluation of PLBCG as a promising vaccine candidate against Mtb.