Transporters regulate trafficking through the biological membrane of living cells and organelles. Therefore, these proteins play an important role in key cellular processes. Obtaining a molecular-level description of the mechanism of transporters is highly desirable to understand and modulate such processes. Different challenges currently complicate this effort, mostly due to transporters' intrinsic properties. They are dynamic and often averse to in vitro characterization. The crossing of the membrane via a transporter depends on both global and local structural changes that will enable substrate binding from one side of the membrane and release on the other. Dedicated approaches are required to monitor these dynamic changes, ideally within the complex membrane environment. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has recently emerged as a powerful biophysical tool to understand transporters' mechanism. This mini-review aims to offer to the reader an overview of the field of HDX-MS applied to transporters. It first summarizes the current workflow for HDX-MS measurements on transporters. It then provides illustrative examples on the molecular insights that are accessible thanks to the technique; following conformational transitions between different states, observing structural changes upon ligand binding and finally understanding the role of lipid-protein interactions.

In the last decades, ligand binding to human TSPO has been largely used in clinical neuroimaging, but little is known about the interaction mechanism. Protein conformational mobility plays a key role in the ligand recognition and both, ligand-free and ligand-bound structures, are mandatory for characterizing the molecular binding mechanism. In the absence of crystals for mammalian TSPO, we have exploited solid-state nuclear magnetic resonance (ssNMR) spectroscopy under magic-angle spinning (MAS) to study the apo form of recombinant mouse TSPO (mTSPO) reconstituted in lipids. This environment has been previously described to permit binding of its high-affinity drug ligand PK11195 and appears therefore favourable for the study of molecular dynamics. We have optimized the physical conditions to get the best resolution for MAS ssNMR spectra of the ligand-free mTSPO. We have compared and combined various ssNMR spectra to get dynamical information either for the lipids or for the mTSPO. Partial assignment of residue types suggests few agreements with the published solution NMR assignment of the PK11195-bound mTSPO in DPC detergent. Moreover, we were able to observe some lateral chains of aromatic residues that were not assigned in solution. 13C double-quantum NMR spectroscopy shows remarkable dynamics for ligand-free mTSPO in lipids which may have significant implications on the recognition of the ligand and/or other protein partners.

The central control of energy homeostasis is a regulatory axis that involves the sensing of nutrients, signaling molecules, adipokines, and neuropeptides by neurons in the metabolic centers of the hypothalamus. However, non-neuronal glial cells are also abundant in the hypothalamus and recent findings have underscored the importance of the metabolic crosstalk and horizontal lipid flux between glia and neurons to the downstream regulation of systemic metabolism. New transgenic models and high-resolution analyses of glial phenotype and function have revealed that glia sit at the nexus between lipid metabolism and neural function, and may markedly impact the brain's response to dietary lipids or the supply of brain-derived lipids. Glia comprise the main cellular compartment involved in lipid synthesis, lipoprotein production, and lipid processing in the brain. In brief, tanycytes provide an interface between peripheral lipids and neurons, astrocytes produce lipoproteins that transport lipids to neurons and other glia, oligodendrocytes use brain-derived and dietary lipids to myelinate axons and influence neuronal function, while microglia can remove unwanted lipids in the brain and contribute to lipid re-utilization through cholesterol efflux. Here, we review recent findings regarding glial-lipid transport and highlight the specific molecular factors necessary for lipid processing in the brain, and how dysregulation of glial-neuronal metabolic crosstalk contributes to imbalanced energy homeostasis. Furthering our understanding of glial lipid metabolism will guide the design of future studies that target horizontal lipid processing in the brain to ameliorate the risk of developing obesity and metabolic disease.

We review the considerable progress during the recent decade in the endeavours of designing, optimising, and utilising carrier particle systems for structural and functional studies of membrane proteins in near-native environments. New and improved systems are constantly emerging, novel studies push the perceived limits of a given carrier system, and specific carrier systems consolidate and entrench themselves as the system of choice for particular classes of target membrane protein systems. This review covers the most frequently used carrier systems for such studies and emphasises similarities and differences between these systems as well as current trends and future directions for the field. Particular interest is devoted to the biophysical properties and membrane mimicking ability of each system and the manner in which this may impact an embedded membrane protein and an eventual structural or functional study.