No abstract

A C-terminal dihydrophobic FL motif plays a vital role in the basolateral targeting of sodium bicarbonate cotransporter 1. To further characterize the role of dihydrophobic FL motif, 1). the FL motif in wild type (PFLS) was reversed to LF (PLFS), 2). the FL motif (PFLS) was shifted upstream (FLPS), and 3). the FL motif (PFLS) was shifted downstream (PSFL). The wild type (PFLS) and its mutant (PLFS) were exclusively expressed on the basolateral membrane by con-focal microscopy, however, the mutant (FLPS) and (PSFL) were predominantly mistargeted to the apical membrane and the cytoplasm, respectively. Functional studies showed that the mutant (PSFL) displayed a remarkably reduced current (p value<0.05 vs wild type). The mutant (PSFL) displayed a more reduced membrane surface expression than the wild type and was co-localized with ER marker. The protein sequence spanning FL motif in kNBC1 C-terminal cytoplasmic tail shows α helical structure, mutants (PLFS) and (PSFL) reduce α-helical contents by circular dichroism study. Reversed FL isn't a constraint for basolateral targeting, but shifting it upstream and downstream are ones. KEYWORDS: Basolateral targeting, mutations with reversed and altered relative position of motif, alpha helical structure.

Due to their activity against different pathogens and low toxicity to host cells, antimicrobial peptides (AMPs) are a promising approach to address the bacterial antimicrobial resistance. Biophysical methods have been used to observe the permeabilizing effect of AMPs on model membranes with the same lipid composition as the bacteria cell wall (BCW). In order to fully understand the antimicrobial properties of these peptides, it also necessary to investigate how AMPs interact with other relevant bacterial cell wall components including peptidoglycan and lipopolysaccharide. One way to better understand AMP interactions with non-lipidic components is to observe their effects on the complete bacterial cell wall. 2 H Nuclear Magnetic Resonance ( 2 H NMR) can be used to measure the orientational order parameters of lipid acyl chain segments. In this work, we have used 2 H NMR to study the effect of AMPs on lipids in the membranes of whole bacteria. We use two techniques to prepare 2 H labeled bacteria with isotope-labeled lipids introduced into the bacterial cell wall. These preparation techniques allow us to use 2 H NMR to compare the orientational order parameter in the bacteria cell wall lipid bilayer of living wild type cells with and without AMP present. The effects two AMPs, either MSI-78 or CAME (Cepropin A (1-8) and melittin (1-10)), on both gramnegative (Escherichia coli) and gram-positive (Bacillus Subtilis) bacteria have been studied. In the presence of both MSI-78 and CAME, the 2 H NMR spectral shape changes in ways that correspond to a decrease in the lipid acyl chain order parameter. 2065-Pos Board B209Selective Membrane Disruption Mechanism of an Antibacterial g-Aapeptide Defined by EPR Spectroscopy . g-AApeptides are a new class of antibacterial peptidomimetics that are not prone to antibiotic resistance and are highly resistant to protease degradation. How g-AApeptides interact with bacterial membranes and alter lipid assembly and properties are unclear, but such information is essential in order to understand their antimicrobial activities. Using electron paramagnetic resonance (EPR) techniques, we characterized the membrane interaction and destabilizing activities of a lipo-cyclic-g-AApeptide. The analyses revealed that the g-AApeptide binding increases the membrane permeability of POPC/POPG liposomes, which mimics negatively-charged bacterial membranes. Moreover, the g-AApeptide interacts strongly with POPC/POPG liposomes, thereby inhibiting membrane fluidity and reducing solvent accessibility around the lipid head group region. Furthermore, binding of the g-AApeptide induces significant lipid-lateral-ordering and membrane thinning. In contrast, minimal membrane property changes were observed upon the g-AApeptide binding for liposomes mimicking mammalian cell membranes, consisting of neutral lipids and cholesterol. Our findings suggest that the g-AApeptide interact and disrupt bacterial membranes through a ''carpet-like'' mechanism. The results illustrated that the intrinsic features of g-AApeptides...