Selective Methyl Labeling of Eukaryotic Membrane Proteins Using Cell-Free Expression

Structural characterization of membrane proteins and other large proteins with NMR relies increasingly on perdeuteration combined with incorporation of specifically protonated amino acid moieties, such as methyl groups of isoleucines, valines, or leucines. The resulting proton dilution reduces dipolar broadening producing sharper resonance lines, ameliorates spectral crowding, and enables measuring of crucial distances between and to methyl groups. While incorporation of specific methyl labeling is now well established for bacterial expression using suitable precursors, corresponding methods are still lacking for cell-free expression, which is often the only choice for producing labeled eukaryotic membrane proteins in mg quantities. Here we show that we can express methyl-labeled human integral membrane proteins cost-effectively by cell-free expression based of crude hydrolyzed ILV-labeled OmpX inclusion bodies. These are obtained in Escherichia coli with very high quantity and represent an optimal intermediate to channel ILV precursors into the eukaryotic proteins

MICA - A new generation of versatile instruments in robotic surgery

Robotic surgery systems are highly complex and expensive pieces of equipment. Demands for lower cost of care can be met if these systems are employable in a flexible manner for a large variety of procedures. To protect the initial investment the capabilities of a robotic system need to be expandable as new tasks arise. To oblige the needs of future robotic support in hospitals, the German Aerospace Center (DLR) has developed the versatile robotic system MiroSurge for medical applications. This paper presents a 3 DoF instrument for Minimally Invasive Robotic Surgery which is mounted to the hollow wrist of the DLR MIRO robot arm. The MICA instrument consists of a versatile drive train and a detachable task specific tool with its tool interface, shaft, 2 DoF wrist, 7 DoF force/torque sensor and the actuated functional end. With the current cabledriven tool, gripping and manipulation forces of above 10 N are feasible and dynamics is high enough for surgery at the beating heart

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape

Comparison of Barley Succession and Take-All Disease as Environmental Factors Shaping the Rhizobacterial Community during Take-All Decline▿

The root disease take-all, caused by Gaeumannomyces graminis var. tritici, can be managed by monoculture-induced take-all decline (TAD). This natural biocontrol mechanism typically occurs after a take-all outbreak and is believed to arise from an enrichment of antagonistic populations in the rhizosphere. However, it is not known whether these changes are induced by the monoculture or by ecological rhizosphere conditions due to a disease outbreak and subsequent attenuation. This question was addressed by comparing the rhizosphere microflora of barley, either inoculated with the pathogen or noninoculated, in a microcosm experiment in five consecutive vegetation cycles. TAD occurred in soil inoculated with the pathogen but not in noninoculated soil. Bacterial community analysis using terminal restriction fragment length polymorphism of 16S rRNA showed pronounced population shifts in the successive vegetation cycles, but pathogen inoculation had little effect. To elucidate rhizobacterial dynamics during TAD development, a 16S rRNA-based taxonomic microarray was used. Actinobacteria were the prevailing indicators in the first vegetation cycle, whereas the third cycle—affected most severely by take-all—was characterized by Proteobacteria, Bacteroidetes, Chloroflexi, Planctomycetes, and Acidobacteria. Indicator taxa for the last cycle (TAD) belonged exclusively to Proteobacteria, including several genera with known biocontrol traits. Our results suggest that TAD involves monoculture-induced enrichment of plant-beneficial taxa

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

Epithelial Cell Line Derived from Endometriotic Lesion Mimics Macrophage Nervous Mechanism of Pain Generation on Proteome and Metabolome Levels

Endometriosis is a benign disease affecting one in ten women of reproductive age worldwide. Although the pain level is not correlated to the extent of the disease, it is still one of the cardinal symptoms strongly affecting the patients’ quality of life. Yet, a molecular mechanism of this pathology, including the formation of pain, remains to be defined. Recent studies have indicated a close interaction between newly generated nerve cells and macrophages, leading to neurogenic inflammation in the pelvic area. In this context, the responsiveness of an endometriotic cell culture model was characterized upon inflammatory stimulation by employing a multi-omics approach, including proteomics, metabolomics and eicosanoid analysis. Differential proteomic profiling of the 12-Z endometriotic cell line treated with TNFα and IL1β unexpectedly showed that the inflammatory stimulation was able to induce a protein signature associated with neuroangiogenesis, specifically including neuropilins (NRP1/2). Untargeted metabolomic profiling in the same setup further revealed that the endometriotic cells were capable of the autonomous production of 7,8-dihydrobiopterin (BH2), 7,8-dihydroneopterin, normetanephrine and epinephrine. These metabolites are related to the development of neuropathic pain and the former three were found up-regulated upon inflammatory stimulation. Additionally, 12-Z cells were found to secrete the mono-oxygenated oxylipin 16-HETE, a known inhibitor of neutrophil aggregation and adhesion. Thus, inflammatory stimulation of endometriotic 12-Z cells led to specific protein and metabolite expression changes suggesting a direct involvement of these epithelial-like cells in endometriosis pain development

Impaired retinal oxygen metabolism and perfusion are accompanied by plasma protein and lipid alterations in recovered COVID-19 patients

Abstract The aim of the present study was to investigate retinal microcirculatory and functional metabolic changes in patients after they had recovered from a moderate to severe acute COVID-19 infection. Retinal perfusion was quantified using laser speckle flowgraphy. Oxygen saturation and retinal calibers were assessed with a dynamic vessel analyzer. Arterio-venous ratio (AVR) was calculated based on retinal vessel diameter data. Blood plasma samples underwent mass spectrometry-based multi-omics profiling, including proteomics, metabolomics and eicosadomics. A total of 40 subjects were included in the present study, of which 29 had recovered from moderate to severe COVID-19 within 2 to 23 weeks before inclusion and 11 had never had COVID-19, as confirmed by antibody testing. Perfusion in retinal vessels was significantly lower in patients (60.6 ± 16.0 a.u.) than in control subjects (76.2 ± 12.1 a.u., p = 0.006). Arterio-venous (AV) difference in oxygen saturation and AVR was significantly lower in patients compared to healthy controls (p = 0.021 for AVR and p = 0.023 for AV difference in oxygen saturation). Molecular profiles demonstrated down-regulation of cell adhesion molecules, NOTCH3 and fatty acids, and suggested a bisphasic dysregulation of nitric oxide synthesis after COVID-19 infection. The results of this study imply that retinal perfusion and oxygen metabolism is still significantly altered in patients well beyond the acute phase of COVID-19. This is also reflected in the molecular profiling analysis of blood plasma, indicating a down-regulation of nitric oxide-related endothelial and immunological cell functions. Trial Registration: ClinicalTrials.gov ( https://clinicaltrials.gov ) NCT05650905