Diversity, Complexity, and Specificity of Bacterial Lipopolysaccharide (LPS) Structures Impacting Their Detection and Quantification.

Endotoxins are toxic lipopolysaccharides (LPSs), extending from the outer membrane of Gram-negative bacteria and notorious for their toxicity and deleterious effects. The comparison of different LPSs, isolated from various Gram-negative bacteria, shows a global similar architecture corresponding to a glycolipid lipid A moiety, a core oligosaccharide, and outermost long O-chain polysaccharides with molecular weights from 2 to 20 kDa. LPSs display high diversity and specificity among genera and species, and each bacterium contains a unique set of LPS structures, constituting its protective external barrier. Some LPSs are not toxic due to their particular structures. Different, well-characterized, and highly purified LPSs were used in this work to determine endotoxin detection rules and identify their impact on the host. Endotoxin detection is a major task to ensure the safety of human health, especially in the pharma and food sectors. Here, we describe the impact of different LPS structures obtained under different bacterial growth conditions on selective LPS detection methods such as LAL, HEK-blue TLR-4, LC-MS2, and MALDI-MS. In these various assays, LPSs were shown to respond differently, mainly attributable to their lipid A structures, their fatty acid numbers and chain lengths, the presence of phosphate groups, and their possible substitutions.

The C-terminal domains of the NMDA receptor: How intrinsically disordered tails affect signalling, plasticity and disease.

NMDA receptors are part of the ionotropic glutamate receptor family, and are crucial for neurotransmission and memory. At the cellular level, the effects of activating these receptors include long-term potentiation (LTP) or depression (LTD). The NMDA receptor is a stringently gated cation channel permeable to Ca2+ , and it shares the molecular architecture of a tetrameric ligand-gated ion channel with the other family members. Its subunits, however, have uniquely long cytoplasmic C-terminal domains (CTDs). While the molecular gymnastics of the extracellular domains have been described in exquisite detail, much less is known about the structure and function of these CTDs. The CTDs vary dramatically in length and sequence between receptor subunits, but they all have a composition characteristic of intrinsically disordered proteins. The CTDs affect channel properties, trafficking and downstream signalling output from the receptor, and these functions are regulated by alternative splicing, protein-protein interactions, and post-translational modifications such as phosphorylation and palmitoylation. Here, we review the roles of the CTDs in synaptic plasticity with a focus on biochemical mechanisms. In total, the CTDs play a multifaceted role as a modifier of channel function, a regulator of cellular location and abundance, and signalling scaffold control the downstream signalling output.

A (2)H magic-angle spinning solid-state NMR characterisation of lipid membranes in intact bacteria.

This work proposes a new approach to characterize cell membranes in intact cells by (2)H solid-state nuclear magnetic resonance (NMR) in only a few hours using magic-angle spinning (MAS) and spectral moment analysis. The method was first validated on model dipalmitoylphosphatidylcholine (DPPC) membranes, allowing the detection of lipid fluctuations below the main transition temperature. Then the lipid dynamics in Escherichia coli membranes was compared in bacteria grown under different diets. More specifically, deuterated palmitic acid was used to isotopically label the phospholipid acyl chains in bacteria membranes, with or without the presence of protonated oleic acid. Our results showed improved lipid fluidity when bacteria were grown in the presence of oleic acid, which helps preserving the natural fatty acid profile in E. coli membranes. The MAS (2)H solid-state NMR study of membranes combined with spectral moment analysis showed to be a fast method compatible with in vivo bacterial studies, and should also be applicable to other micro-organisms to obtain molecular information on living cells by solid-state NMR.

In-Cell Solid-State NMR: An Emerging Technique for the Study of Biological Membranes.

Biological molecular processes are often studied in model systems, which simplifies their inherent complexity but may cause investigators to lose sight of the effects of the molecular environment. Information obtained in this way must therefore be validated by experiments in the cell. NMR has been used to study biological cells since the early days of its development. The first NMR structural studies of a protein inside a cell (by solution-state NMR) and of a membrane protein (by solid-state NMR) were published in 2001 and 2011, respectively. More recently, dynamic nuclear polarization, which has been used to enhance the signal in solid-state NMR, has also been applied to the study of frozen cells. Much progress has been made in the past 5 years, and in this review we take stock of this new technique, which is particularly appropriate for the study of biological membranes.