Membrane Lipid Environment Research Articles

Modulation of GPCR Rhodopsin Function by Membrane Lipids and Water

G protein‐coupled receptors (GPCRs) are responsible for the transduction of signals across lipid membranes and are the largest family of targets (~35%) for currently approved drugs. They exist as dynamic conformational ensembles with multiple inactive and active conformational substates described by an energy landscape model. Consequently, understanding the modulation of conformational dynamics of GPCRs by soft matter such as membrane lipids and cellular water in the receptor’s environment is critical to understanding GPCR efficacy and selectivity. Here we show how membrane lipids and cellular water play crucial roles in shaping the energy landscape of GPCR activation, thus acting as allosteric modulators. We investigated ways in which the receptor hydration level and lipid bilayer composition influence the metarhodopsin equilibrium of the archetypical GPCR rhodopsin in native and POPC recombinant membranes after light activation. Using different polyethylene glycol (PEG) solutions, the osmotic pressure on rhodopsin was varied, while shifting of the metarhodopsin equilibrium was probed using UV‐Visible spectroscopy. Our results show a flood of ~80‐100 water molecules into the rhodopsin interior during photoactivation, forming a solvent‐swollen Meta‐II active state. Dehydrating conditions favor Meta‐I through the efflux of water from the protein interior yet simultaneously increase bilayer thickness, which should favor Meta‐II. Because the osmotic effect on the protein is more significant than the effect of the lipid bilayer, the overall equilibrium is generally shifted to Meta‐I. However, small osmolytes favored the Meta‐II state because they can penetrate the protein core, decreasing the osmotic effect on the protein. Furthermore, the metarhodopsin equilibrium was shifted towards the Meta‐I state in POPC recombinant membranes compared to the native membrane environment. Analysis of transducin C‐terminal peptide‐binding isotherms revealed that the G‐prottein binding affinity is significantly decreased when the lipid environment is changed from the native lipids to POPC lipids. The POPC lipid membrane has zero spontaneous curvature that shifts the equilibrium towards the more compact, inactive Meta‐I state. By contrast, the native lipid membrane environment possesses a negative spontaneous curvature that favors the more expanded state of Meta‐II. Our results delineate the crucial role of soft matter in regulating the metarhodopsin equilibrium in a membrane environment.

Protonation of model ionizable sidechains in transmembrane protein domains

The ionization states of individual amino acid residues of membrane proteins in lipid membrane environment are difficult to decipher or assign directly. The effective pK(a) values of protein groups are determined by a complex interplay between local polarity, Coulomb interactions, and structural reorganizations. The analysis is further complicated by the dearth of information about gradients in polarity, electric potentials, and hydration at the protein-membrane interface. In this work we report on a spin-labeling EPR method for assessing effects of membrane surface potential, local environment at the protein-membrane interface, and water penetration along this interface on effective pK(a) of membrane-buried ionizable groups.

All-Atom Modeling of Complex Cellular Membranes.

Cell membranes are composed of a variety of lipids and proteins where they interact with each other to fulfill their roles. The first step in modeling these interactions in molecular simulations is to have reliable mimetics of the membrane's lipid environment. This Feature Article presents our recent efforts to model complex cellular membranes using all-atom force fields. A short review of the CHARMM36 (C36) lipid force field and its recent update to incorporate the long-range dispersion is presented. Key examples of model membranes mimicking various species and organelles are given. These include single-celled organisms such as bacteria (E. coli., chlamydia, and P. aeruginosa) and yeast (plasma membrane, endoplasmic reticulum, and trans-Golgi network) and more advanced ones such as plants (soybean and Arabidopsis thaliana) and mammals (ocular lens, stratum corneum, and peripheral nerve myelin). Leaflet asymmetry in composition has also been applied to some of these models. With the increased lipid diversity in the C36 lipid FF, these complex models can better reflect the structural, mechanical, and dynamic properties of realistic membranes and open an opportunity to study biological processes involving other molecules.

Low light acclimation strategy of the brown macroalga Undaria pinnatifida: Significance of lipid and fatty acid remodeling for photosynthetic competence.

Brown macroalgae, being important components of benthic communities in temperate regions, are frequently subjected to light limitation. To extend our understanding of their low light acclimation strategies to the regulation of membrane lipid environment, photosynthetic characteristics, lipid class, fatty acid profiles and chloroplast ultrastructure were compared in Undaria pinnatifida (Phaeophyceae, Ochrophyta) after long-term exposure to low and moderate light intensities (LL, 100 and ML, 280 µmol photons · m-2 · s-1 ). We show that light limitation significantly increased PSII quantum efficiency and photosynthetic electron transport rate, enhanced pigment contents and concentration of thylakoid membranes in chloroplasts but decreased the distance between the thylakoid stacks. These physiological alterations at LL were accompanied by a selective remodeling of thylakoid membrane lipids driven by increases in monogalactosyldiacylglycerol (MGDG) and phosphatidylglycerol (PG) contents. Light limitation also induced active production of PG specific trans-Δ3 -hexadecenoic acid and accumulation of n-3 polyunsaturated fatty acids (PUFA) mostly in PG and MGDG at the expense of the rise in 18:3n-3 and 20:5n-3, 18:4n-3, respectively. These changes in lipid and FA profiles are apparently responsible for supporting thylakoid biogenesis and efficient photosynthesis at light limitation, thus contributing to photoacclimation strategies in brown algae. The content of triacylglycerols (TAG) and the level of their PUFA were decreased at LL, suggesting the consumption of TAG as a source of PUFA and energy reserves. Thus, U. pinnatifida is able to successfully overcome periods of low irradiance through the effective light harvesting and utilization that are provided by high flexibility of lipid biosynthesis.

Gangliosides smelt nanostructured amyloid Aβ(1–40) fibrils in a membrane lipid environment

Gangliosides induced a smelting process in nanostructured amyloid fibril-like films throughout the surface properties contributed by glycosphingolipids when mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC)/Aβ(1–40) amyloid peptide. We observed a dynamical smelting process when pre-formed amyloid/phospholipid mixture is laterally mixed with gangliosides. This particular environment, gangliosides/phospholipid/Aβ(1–40) peptide mixed interfaces, showed complex miscibility behavior depending on gangliosides content. At 0% of ganglioside covered surface respect to POPC, Aβ(1–40) peptide forms fibril-like structure. In between 5 and 15% of gangliosides, the fibrils dissolve into irregular domains and they disappear when the proportion of gangliosides reach the 20%. The amyloid interfacial dissolving effect of gangliosides is taken place at lateral pressure equivalent to the organization of biological membranes.Domains formed at the interface are clearly evidenced by Brewster Angle Microscopy and Atomic Force Microscopy when the films are transferred onto a mica support. The domains are thioflavin T (ThT) positive when observed by fluorescence microscopy.We postulated that the smelting process of amyloids fibrils-like structure at the membrane surface provoked by gangliosides is a direct result of a new interfacial environment imposed by the complex glycosphingolipids. We add experimental evidence, for the first time, how a change in the lipid environment (increase in ganglioside proportion) induces a rapid loss of the asymmetric structure of amyloid fibrils by a simple modification of the membrane condition (a more physiological situation).

TOAC spin-labeled peptides tailored for DNP-NMR studies in lipid membrane environments

The benefit of combining in-cell solid-state dynamic nuclear polarization (DNP) NMR and cryogenic temperatures is providing sufficient signal/noise and preservation of bacterial integrity via cryoprotection to enable in situ biophysical studies of antimicrobial peptides. The radical source required for DNP was delivered into cells by adding a nitroxide-tagged peptide based on the antimicrobial peptide maculatin 1.1 (Mac1). In this study, the structure, localization, and signal enhancement properties of a single (T-MacW) and double (T-T-MacW) TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin-labeled Mac1 analogs were determined within micelles or lipid vesicles. The solution NMR and circular dichroism results showed that the spin-labeled peptides adopted helical structures in contact with micelles. The peptides behaved as an isolated radical source in the presence of multilamellar vesicles, and the electron paramagnetic resonance (EPR) electron-electron distance for the doubly spin-labeled peptide was ∼1 nm. The strongest paramagnetic relaxation enhancement (PRE) was observed for the lipid NMR signals near the glycerol-carbonyl backbone and was stronger for the doubly spin-labeled peptide. Molecular dynamics simulation of the T-T-MacW radical source in phospholipid bilayers supported the EPR and PRE observations while providing further structural insights. Overall, the T-T-MacW peptide achieved better 13C and 15N signal NMR enhancements and 1H spin-lattice T1 relaxation than T-MacW.

Illuminating Disorder Induced by Glu in a Stable Arg-Anchored Transmembrane Helix.

Membrane proteins are vital for biological function and are complex to study. Even in model peptide-lipid systems, the combined influence or interaction of pairs of chemical groups still is not well understood. Disordered proteins, whether in solution or near lipid membranes, are an emerging paradigm for the initiation and control of biological function. The disorder can involve molecular orientation as well as molecular folding. This paper reports an astonishing induction of disorder when one Glu residue is introduced into a highly stable 23-residue transmembrane helix. The parent helix is anchored by a single Arg residue, tilted at a well-defined angle with respect to the DOPC bilayer normal and undergoes rapid cone precession. When Glu is introduced two residues away from Arg, with 200° (or 160°) radial separation, the helix properties change radically to exhibit a multiplicity of three or more disordered states. The helix characteristics have been monitored by deuterium (2H) NMR spectroscopy as functions of the pH and lipid bilayer composition. The disordered multistate behavior of the (Glu, Arg)-containing helix varies with the lipid bilayer thickness and pH. The results highlight a fundamental induction of protein multistate properties by a single Glu residue in a lipid membrane environment.

Modulation of adenosine A2a receptor oligomerization by receptor activation and PIP2 interactions

SummaryGPCRs have been shown to form oligomers, which generate distinctive signaling outcomes. However, the structural nature of the oligomerization process remains uncertain. We have characterized oligomeric configurations of the adenosine A2a receptor (A2aR) by combining large-scale molecular dynamics simulations with Markov state models. These oligomeric structures may also serve as templates for studying oligomerization of other class A GPCRs. Our simulation data revealed that receptor activation results in enhanced oligomerization, more diverse oligomer populations, and a more connected oligomerization network. The active state conformation of the A2aR shifts protein-protein association interfaces to those involving intracellular loop ICL3 and transmembrane helix TM6. Binding of PIP2 to A2aR stabilizes protein-protein interactions via PIP2-mediated association interfaces. These results indicate that A2aR oligomerization is responsive to the local membrane lipid environment. This, in turn, suggests a modulatory effect on A2aR whereby a given oligomerization profile favors the dynamic formation of specific supramolecular signaling complexes.

Vitamin B12 Inhibits Aβ Fibrillation and Disaggregates Preformed Fibrils in the Presence of Synthetic Neuronal Membranes.

The aggregation of amyloid β (Aβ) peptide with subsequent formation of fibrils which deposit in senile plaques is considered one of the key triggers of Alzheimer's disease (AD). Molecules targeting the inhibition of Aβ fibrillation and/or the disruption of Aβ fibrils are thus promising approaches for the medical prevention and treatment of AD. However, amyloid formation is a complex process strongly influenced by the cellular environment, such as cell membranes, which may affect the effectiveness of therapeutic molecules. In this study, the effect of the vitamin B12 (VB12) on the formation and disaggregation of Aβ1-42 fibrils was investigated in the presence of artificial neuronal membranes mimicked by liposomes. Evidence showed that VB12 slows down the Aβ fibrillization and reduces the content of fibrils in aqueous solution. Moreover, the vitamin exhibited a strong ability to disrupt preformed fibrils. However, the presence of lipid vesicles compromised the VB12's antiamyloidogenic properties due to the competitive interaction of the vitamin with the lipid membrane and the Aβ peptide. Even so, VB12 was effective in inhibiting the fibril formation and disaggregating fibrils in the lipid membrane environment. Thereby, these results indicate that VB12 could be a promising molecule both for the prevention and cure of AD, thus warranting its study in animal models.

Detergent-free systems for structural studies of membrane proteins.

Membrane proteins play vital roles in living organisms, serving as targets for most currently prescribed drugs. Membrane protein structural biology aims to provide accurate structural information to understand their mechanisms of action. The advance of membrane protein structural biology has primarily relied on detergent-based methods over the past several decades. However, detergent-based approaches have significant drawbacks because detergents often damage the native protein-lipid interactions, which are often crucial for maintaining the natural structure and function of membrane proteins. Detergent-free methods recently have emerged as alternatives with a great promise, e.g. for high-resolution structure determinations of membrane proteins in their native cell membrane lipid environments. This minireview critically examines the current status of detergent-free methods by a comparative analysis of five groups of membrane protein structures determined using detergent-free and detergent-based methods. This analysis reveals that current detergent-free systems, such as the styrene-maleic acid lipid particles (SMALP), the diisobutyl maleic acid lipid particles (DIBMALP), and the cycloalkane-modified amphiphile polymer (CyclAPol) technologies are not better than detergent-based approaches in terms of maintenance of native cell membrane lipids on the transmembrane domain and high-resolution structure determination. However, another detergent-free technology, the native cell membrane nanoparticles (NCMN) system, demonstrated improved maintenance of native cell membrane lipids with the studied membrane proteins, and produced particles that were suitable for high-resolution structural analysis. The ongoing development of new membrane-active polymers and their optimization will facilitate the maturation of these new detergent-free systems.

Acute and chronic effects of temperature on membrane adjustments in the gills of a neotropical catfish.

Structural modifications in the gill membranes maintain homeostasis under the influence of temperature changes. We hypothesized that thermal acclimation would result in significant modification of phospholipid fatty acids, with modulation of sodium pump activity during acute (24 and 48h) and chronic (15days) thermal shifts in the neotropical reophilic catfish Steindachneridion parahybae. Indeed, the time-course experiment showed acute and chronic changes in gill membrane at the lowest temperatures, notably linked to maintenance of membrane fluidity: significant preferential changes in phosphatidylethanolamine, with decrease of saturated fatty acids and increase of C18:1 in all groups kept below 30°C in chronic trial, increase in polyunsaturated fatty acids n6 and C18:1 at 17 and 12°C compared to 24°C, as soon as the temperature was changed (initial time). Additionally, the activity of the sodium pump increased at 12°C, but without apparent connection with the altered lipid environment. The animals maintained at the lowest temperature showed a higher mortality, possibly because of the approach to the minimum critical temperature for this species, and unexpected results of changes in the fatty acid profile, such as decreased docosahexaenoic acid in phosphatidylethanolamine and increased saturated fatty acids in phosphatidylcholine. This set of mechanisms highlights rheostatic adjustments in this species in the face of temperature changes.

Membrane Lipids and Cellular Water Modulate the G‐Protein–Coupled Receptor Activation

G-protein-coupled receptors (GPCRs) are a pivotal superfamily of seven-helix transmembrane receptors responsible for the transduction of stimuli across cellular membranes. They are the targets of ~30% of the pharmaceuticals currently in the market. Understanding how the soft membrane matter (membrane Lipids and Cellular Water) influences the structural changes during the activation of GPCRs is crucial to discovering and developing novel GPCR-targeted drugs. Their dynamic conformational ensembles encompassing various inactive and active states can be biased by the lipids and surrounding aqueous environments [1]. By using different polyethylene glycol (PEG) solutions, we explored the effect of osmotic pressure and lipid bilayer composition on the metarhodopsin equilibrium of the archetypical GPCR rhodopsin in native membranes and POPC recombinant membranes [2]. Our results show a flood of ~80 water molecules into the rhodopsin interior during photoactivation, forming a solvent-swollen Meta-II active state [3]. Under applied osmotic pressure, the overall equilibrium generally shifted to Meta-I. Dehydrating conditions favor Meta-I through the efflux of water from the protein interior while increasing bilayer thickness and the monolayer spontaneous curvature favor Meta-II. The osmotic effect on the protein is more significant than the effect of the lipid bilayer. However, small osmolytes favored the Meta-II state at lower concentrations because they can penetrate the protein core giving a lower excluded volume, decreasing the osmotic effect on the protein and favoring the Meta-II state. Furthermore, the metarhodopsin equilibrium was shifted towards the Meta-I state in POPC recombinant membranes compared to the native membrane environment. Analysis of transducin C-terminal peptide-binding isotherms revealed that the binding affinity is significantly decreased when the lipid environment is changed from the native lipids to POPC lipids. The POPC lipid membrane has zero-spontaneous curvature that shifts the equilibrium towards the more compact, inactive Meta-I state. By contrast, the native lipid membrane environment has a negative spontaneous curvature that favors the more expanded state of Meta-II. Our results delineate the crucial role of soft matter in regulating the metarhodopsin equilibrium in a membrane environment. [1] M.F. Brown (2017) Annu. Rev. Biophys. 46, 379-410. [2] N. Weerasinghe et al. (2018) Biophys. J. 114, 274a. [3] U. Chawla et al. (2020) Angew. Chem. Int. Ed. doi: https://doi.org/10.1002/ange.202003342.

Caveolin-1-driven membrane remodelling regulates hnRNPK-mediated exosomal microRNA sorting in cancer.

BackgroundCaveolae proteins play diverse roles in cancer development and progression. In prostate cancer, non‐caveolar caveolin‐1 (CAV1) promotes metastasis, while CAVIN1 attenuates CAV1‐induced metastasis. Here, we unveil a novel mechanism linking CAV1 to selective loading of exosomes with metastasis‐promoting microRNAs.ResultsWe identify hnRNPK as a CAV1‐regulated microRNA binding protein. In the absence of CAVIN1, non‐caveolar CAV1 drives localisation of hnRPNK to multi‐vesicular bodies (MVBs), recruiting AsUGnA motif‐containing miRNAs and causing their release within exosomes. This process is dependent on the lipid environment of membranes as shown by cholesterol depletion using methyl‐β‐cyclodextrin or by treatment with n‐3 polyunsaturated fatty acids. Consistent with a role in bone metastasis, knockdown of hnRNPK in prostate cancer PC3 cells abolished the ability of PC3 extracellular vesicles (EV) to induce osteoclastogenesis, and biofluid EV hnRNPK is elevated in metastatic prostate and colorectal cancer.ConclusionsTaken together, these results support a novel pan‐cancer mechanism for CAV1‐driven exosomal release of hnRNPK and associated miRNA in metastasis, which is modulated by the membrane lipid environment.

Mechanisms of mitochondrial cell death.

Mitochondria are double-membrane bound organelles that not only provide energy for intracellular metabolism, but also play a key role in the regulation of cell death. Mitochondrial outer membrane permeabilization (MOMP), allowing the release of intermembrane space proteins like cytochrome c, is considered a point of no return in apoptosis. MOMP is controlled by the proteins of the B-cell lymphoma 2 (BCL-2) family, including pro-and anti-apoptotic members, whose balance determines the decision between cell death and survival. Other factors such as membrane lipid environment, membrane dynamics, and inter-organelle communications are also known to influence this process. MOMP and apoptosis have been acknowledged as immunologically silent. Remarkably, a growing body of evidence indicates that MOMP can engage in various pro-inflammatory signaling functions. In this mini-review, we discuss about our current knowledge on the mechanisms of mitochondrial apoptosis, as well as the involvement of mitochondria in other kinds of programmed cell death pathways.

Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs.

G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1β1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.

Single-Chain Dimensions of an Archetypal Phase-Separating Intrinsically Disordered Protein Region

Membrane-less organelles (MLOs) are non-stoichiometric assemblies of concentrated biomolecules lacking a surrounding lipid membrane and form via liquid-liquid phase separation (LLPS). They facilitate a variety of cellular processes, and the dysregulation of phase separation can lead to debilitating diseases. Intrinsically disordered regions (IDRs) in proteins have often been found to drive phase separation. Due to lack of structure, disordered proteins are challenging to study with traditional structural biology methods, and this combined with crowded dense phase environments requires careful experimental design in order to extract quantitative information. We are directing our studies at an archetypal IDR that undergoes homotypic phase separation, i.e. the prion-like domain (PLD) of hnRNPA1. Here we use single-molecule fluorescence techniques to explore the dimensions and dynamics of hnRNPA1 in light and dense phases. We characterize single-chain behavior in response to salt concentration, which is used to induce phase separation, and elucidate how this is modulated by sequence variation. Initial measurements show that the PLD is a compact disordered protein in the dilute regime and that modulation of the driving force for phase separation also modulates the single-chain dimensions. This work will provide insights into structural and dynamic features of phase-separating biomolecules and will enable the investigation of maturation effects responsible for disease states.

Solvation Drives G-Protein-Coupled Receptor Activation

G-protein–coupled receptors (GPCRs) are membrane proteins that transduce information across lipid bilayers. Their dynamic conformational ensembles encompassing various inactive and active states can be biased by the lipids and surrounding aqueous environments. By using different polyethylene glycol (PEG) solutions, we explored the effect of osmotic pressure and lipid bilayer composition on the metarhodopsin equilibrium of the archetypical GPCR rhodopsin in native membranes and POPC recombinant membranes. Our results show a flood of ∼80 water molecules into the rhodopsin interior during photoactivation, forming a solvent-swollen Meta-II active state [1]. Under applied osmotic pressure, the overall equilibrium generally shifted to Meta-I. Dehydrating conditions favor Meta-I through an efflux of water from the protein interior, while increasing bilayer thickness and the monolayer spontaneous curvature favor Meta-II. The osmotic effect on the protein is more significant than the effect of the lipid bilayer. However, small osmolytes favored the Meta-II state at lower concentrations, because they can penetrate the protein core giving a lower excluded volume, decreasing the osmotic effect on the protein and favoring the Meta-II state. Furthermore, the metarhodopsin equilibrium was shifted towards the Meta-I state in POPC recombinant membranes compared to the native membrane environment. Analysis of transducin C-terminal peptide-binding isotherms revealed that the binding affinity is significantly decreased when the lipid environment is changed from the native lipids to POPC lipids. The POPC lipid membrane has zero-spontaneous curvature that shifts the equilibrium towards the more compact, inactive Meta-I state. By contrast, the native lipid membrane environment has a negative spontaneous curvature that favors the more expanded state of Meta-II. Our results delineate the crucial role of soft matter in regulating the metarhodopsin equilibrium in a membrane environment. [1] U. Chawla et al. (2020) Angew. Chem. Int. Ed. DOI: https://doi.org/10.1002/ange.202003342.

Characterization, Docking and Molecular Dynamics Simulation of Gonadotropin-Inhibitory Hormone Receptor (GnIHR2) in Labeo Catla.

GnIH receptors (GnIHRs) belong to the family of G-protein coupled receptors (GPCRs) and play a key role in the regulation of reproduction from fishes to mammals, either by inhibiting or stimulating the expression of gonadotropins. The aim of this study was to characterize GnIH receptor (GnIHR2) from Indian Major Carp, Labeo catla and its docking and simulation with GnIH antagonist RF313. The full length sequence of GnIHR2 was obtained with RACE PCR. The docking analysis of RF313 with GnIHR2 receptor was performed with AutoDock v. 4.2.6 and molecular dynamics (MD) simulation with GROMACS 5.0. In the present study, we cloned full-length cDNA (1733 bp) of GnIHR2 from the brain of L. catla. The phylogenetic analysis showed clustering of catla GnIHR2 with goldfish and zebrafish in the GPR147 group. L. catla GnIHR2 receptor comprised seven transmembrane domains and the 3D-structure was predicted by I-TASSER tool. The docking analysis revealed high binding affinity (-11.6 kcal/mol) of GnIH antagonist, RF313 towards GnIHR2 receptor. The primary bonds involved were alkyl and hydrogen bonds while the amino acids participated were proline 43, 210, 339, cysteine 214, leucine 211, serine 213 and phenylalanine 338. The MD simulation analysis of docked complex for 100 nano-seconds (ns) in the lipid membrane environment showed the stability of the complex with time. Our study showed that GnIH antagonist, RF313 interact tightly with the GnIH receptor, GnIHR2 of L. catla. To the best of our knowledge, this is the first report on computational modelling and MD simulation of GnIH receptor in fishes. This will help in functional characterization studies of GnIH/GnIHR system in vertebrates.

Capturing Membrane Protein Ribosome Nascent Chain Complexes in a Native-like Environment for Co-translational Studies.

Co-translational folding studies of membrane proteins lag behind cytosolic protein investigations largely due to the technical difficulty in maintaining membrane lipid environments for correct protein folding. Stalled ribosome-bound nascent chain complexes (RNCs) can give snapshots of a nascent protein chain as it emerges from the ribosome during biosynthesis. Here, we demonstrate how SecM-facilitated nascent chain stalling and native nanodisc technologies can be exploited to capture in vivo-generated membrane protein RNCs within their native lipid compositions. We reveal that a polytopic membrane protein can be successfully stalled at various stages during its synthesis and the resulting RNC extracted within either detergent micelles or diisobutylene–maleic acid co-polymer native nanodiscs. Our approaches offer tractable solutions for the structural and biophysical interrogation of nascent membrane proteins of specified lengths, as the elongating nascent chain emerges from the ribosome and inserts into its native lipid milieu.

Gating of human TRPV3 in a lipid bilayer.

The TRPV3 channel plays a critical role in skin physiology, and mutations in TRPV3 result in the development of a congenital skin disorder, Olmsted syndrome. Here we describe multiple cryo-electron microscopy structures of human TRPV3 reconstituted into lipid nanodiscs, representing distinct functional states during the gating cycle. The ligand-free, closed conformation reveals well-ordered lipids interacting with the channel and two physical constrictions along the ion conduction pore involving both the extracellular selectivity filter and intracellular helix bundle crossing. Both the selectivity filter and bundle crossing expand upon activation, accompanied by substantial structural rearrangements at the cytoplasmic inter-subunit interface. Transition to the inactivated state involves a secondary structure change of the pore-lining helix, which contains a π-helical segment in the closed and open conformations but becomes entirely α-helical upon inactivation. Together with electrophysiological characterization, structures of TRPV3 in a lipid membrane environment provide unique insights into channel activation and inactivation mechanisms.