Mechanism of membrane-curvature generation by ER-tubule shaping proteins.

The endoplasmic reticulum (ER) network consists of tubules with high membrane curvature in cross-section, generated by the reticulons and REEPs. These proteins have two pairs of trans-membrane (TM) segments, followed by an amphipathic helix (APH), but how they induce curvature is poorly understood. Here, we show that REEPs form homodimers by interaction within the membrane.

Improved strategy for isoleucine H/C methyl labeling in Pichia pastoris.

Site specific methyl labeling combined with methyl TROSY offers a powerful NMR approach to study structure and dynamics of proteins and protein complexes of high molecular weight. Robust and cost-effective methods have been developed for site specific protein H/C methyl labeling in an otherwise deuterated background in bacteria. However, bacterial systems are not suitable for expression and isotope labeling of many eukaryotic and membrane proteins.

Isotopic Labeling of Eukaryotic Membrane Proteins for NMR Studies of Interactions and Dynamics.

Membrane proteins, and especially G-protein coupled receptors (GPCRs), are increasingly important targets of structural biology studies due to their involvement in many biomedically critical pathways in humans. These proteins are often highly dynamic and thus benefit from studies by NMR spectroscopy in parallel with complementary crystallographic and cryo-EM analyses. However, such studies are often complicated by a range of practical concerns, including challenges in preparing suitably isotopically labeled membrane protein samples, large sizes of protein/detergent or protein/lipid complexes, and limitations on sample concentrations and stabilities.

Crystal structure of the human NK tachykinin receptor.

The NK tachykinin G-protein-coupled receptor (GPCR) binds substance P, the first neuropeptide to be discovered in mammals. Through activation of NKR, substance P modulates a wide variety of physiological and disease processes including nociception, inflammation, and depression. Human NKR (hNKR) modulators have shown promise in clinical trials for migraine, depression, and emesis.

Ligand modulation of sidechain dynamics in a wild-type human GPCR.

GPCRs regulate all aspects of human physiology, and biophysical studies have deepened our understanding of GPCR conformational regulation by different ligands. Yet there is no experimental evidence for how sidechain dynamics control allosteric transitions between GPCR conformations. To address this deficit, we generated samples of a wild-type GPCR (AR) that are deuterated apart from H/C NMR probes at isoleucine δ1 methyl groups, which facilitated H/C methyl TROSY NMR measurements with opposing ligands.