Search

G‐Protein‐gekoppelte Rezeptoren initiieren die Signaltransduktion nach Ligandenbindung. Gegenstand der Studie ist der Wachstumshormon‐Sekreteagogum‐Rezeptor (GHSR), der durch das Peptid Ghrelin aktiviert wird. Obwohl es Strukturen des GHSR in verschiedenen Stadien der Aktivierung gibt, wurde die molekulare Dynamik dieser Zustände bisher nicht beschrieben. Wir analysierten die Trajektorien von Molekulardynamiksimulationen mittels Detektoranalyse, um die Dynamik des Apo‐ und des Liganden‐gebundenen Zustands zu vergleichen. Diese Analyse liefert zeitskalenspezifische Bewegungsamplituden. Wir identifizierten Unterschiede in der Dynamik des Apo‐ und Ghrelin‐gebundenen Zustands, insbesondere in der extrazellulären Schleife 2 und den Transmembranhelices 5–7. Kernresonanz‐Spektren der Histidinreste im GHSR zeigten Unterschiede in den chemischen Verschiebungen dieser Regionen. Wir evaluierten zeitskalenspezifische Korrelationen der Bewegungen zwischen Ligand und Rezeptor und fanden Korrelationen zwischen den N‐terminalen Ghrelin‐Resten und dem Rezeptor, nicht aber für den C‐Terminus von Ghrelin. Eine Hauptkomponentenanalyse lieferte Rückschlüsse auf die Energielandschaft des GHSR. [ABSTRACT FROM AUTHOR]

Mass spectrometry (MS)‐based proteomics provides unprecedented opportunities for understanding the structure and function of proteins in complex biological systems; however, protein solubility and sample preparation before MS remain a bottleneck preventing high‐throughput proteomics. Herein, we report a high‐throughput bottom‐up proteomic method enabled by a newly developed MS‐compatible photocleavable surfactant, 4‐hexylphenylazosulfonate (Azo) that facilitates robust protein extraction, rapid enzymatic digestion (30 min compared to overnight), and subsequent MS‐analysis following UV degradation. Moreover, we developed an Azo‐aided bottom‐up method for analysis of integral membrane proteins, which are key drug targets and are generally underrepresented in global proteomic studies. Furthermore, we demonstrated the ability of Azo to serve as an "all‐in‐one" MS‐compatible surfactant for both top‐down and bottom‐up proteomics, with streamlined workflows for high‐throughput proteomics amenable to clinical applications. [ABSTRACT FROM AUTHOR]

The preparation of native S‐palmitoylated (S‐palm) membrane proteins is one of the unsolved challenges in chemical protein synthesis. Herein, we report the first chemical synthesis of S‐palm membrane proteins by removable‐backbone‐modification‐assisted Ser/Thr ligation (RBMGABA‐assisted STL). This method involves two critical steps: 1) synthesis of S‐palm peptides by a new γ‐aminobutyric acid based RBM (RBMGABA) strategy, and 2) ligation of the S‐palm RBM‐modified peptides to give the desired S‐palm product by the STL method. The utility of the RBMGABA‐assisted STL method was demonstrated by the synthesis of rabbit S‐palm sarcolipin (SLN) and S‐palm matrix‐2 (M2) ion channel. The synthesis of S‐palm membrane proteins highlights the importance of developing non‐NCL methods for chemical protein synthesis. [ABSTRACT FROM AUTHOR]

Atherosclerosis (AS) is a major contributor to cardiovascular diseases worldwide, and alleviating inflammation is a promising strategy for AS treatment. Here, we report molecularly engineered M2 macrophage‐derived exosomes (M2 Exo) with inflammation‐tropism and anti‐inflammatory capabilities for AS imaging and therapy. M2 Exo are derived from M2 macrophages and further electroporated with FDA‐approved hexyl 5‐aminolevulinate hydrochloride (HAL). After systematic administration, the engineered M2 Exo exhibit excellent inflammation‐tropism and anti‐inflammation effects via the surface‐bonded chemokine receptors and the anti‐inflammatory cytokines released from the anti‐inflammatory M2 macrophages. Moreover, the encapsulated HAL can undergo intrinsic biosynthesis and metabolism of heme to generate anti‐inflammatory carbon monoxide and bilirubin, which further enhance the anti‐inflammation effects and finally alleviate AS. Meanwhile, the intermediate protoporphyrin IX (PpIX) of the heme biosynthesis pathway permits the fluorescence imaging and tracking of AS. [ABSTRACT FROM AUTHOR]

In oriented‐sample (OS) solid‐state NMR of membrane proteins, the angular‐dependent dipolar couplings and chemical shifts provide a direct input for structure calculations. However, so far only 1H–15N dipolar couplings and 15N chemical shifts have been routinely assessed in oriented 15N‐labeled samples. The main obstacle for extending this technique to membrane proteins of arbitrary topology has remained in the lack of additional experimental restraints. We have developed a new experimental triple‐resonance NMR technique, which was applied to uniformly doubly (15N, 13C)‐labeled Pf1 coat protein in magnetically aligned DMPC/DHPC bicelles. The previously inaccessible 1Hα–13Cα dipolar couplings have been measured, which make it possible to determine the torsion angles between the peptide planes without assuming α‐helical structure a priori. The fitting of three angular restraints per peptide plane and filtering by Rosetta scoring functions has yielded a consensus α‐helical transmembrane structure for Pf1 protein. [ABSTRACT FROM AUTHOR]

The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important, but poorly resolved, are the forces present during the initial unfolding of a membrane protein, where the most native set of interactions is present. A high‐precision, atomic force microscopy assay was developed to study the initial unfolding of bacteriorhodopsin. A rapid near‐equilibrium folding between the first three unfolding states was discovered, the two transitions corresponded to the unfolding of five and three amino acids, respectively, when using a cantilever optimized for 2 μs resolution. The third of these states was retinal‐stabilized and previously undetected, despite being the most mechanically stable state in the whole unfolding pathway, supporting 150 pN for more than 1 min. This ability to measure the dynamics of the initial unfolding of bacteriorhodopsin provides a platform for quantifying the energetics of membrane proteins under native‐like conditions. [ABSTRACT FROM AUTHOR]

Regulation of protein activity is essential for revealing the molecular mechanisms of biological processes. DNA and RNA achieve many uniquely efficient functions, such as genetic expression and regulation. The chemical capability to synthesize artificial nucleotides can expand the chemical space of nucleic acid libraries and further increase the functional diversity of nucleic acids. Herein, a versatile method has been developed for modular expansion of the chemical space of nucleic acid libraries, thus enabling the generation of aptamers able to regulate protein activity. Specifically, an aptamer that targets integrin alpha3 was identified and this aptamer can inhibit cell adhesion and migration. Overall, this chemical‐design‐assisted in vitro selection approach enables the generation of functional nucleic acids for elucidating the molecular basis of biological activities and uncovering a novel basis for the rational design of new protein‐inhibitor pharmaceuticals. [ABSTRACT FROM AUTHOR]

Exosomes are small (30–100 nm) membrane vesicles that serve as regulatory agents for intercellular communication in cancers. Currently, exosomes are detected by immuno‐based assays with appropriate pretreatments like ultracentrifugation and are time consuming (>12 h). We present a novel pretreatment‐free fluorescence‐based sensing platform for intact exosomes, wherein exchangeable antibodies and fluorescent reporter molecules were aligned inside exosome‐binding cavities. Such antibody‐containing fluorescent reporter‐grafted nanocavities were prepared on a substrate by well‐designed molecular imprinting and post‐imprinting modifications to introduce antibodies and fluorescent reporter molecules only inside the binding nanocavities, enabling sufficiently high sensitivity to detect intact exosomes without pretreatment. The effectiveness of the system was demonstrated by using it to discriminate between normal exosomes and those originating from prostate cancer and analyze exosomes in tear drops. [ABSTRACT FROM AUTHOR]

Abstract: To tackle the problems associated with membrane protein (MP) instability in detergent solutions, we designed a series of glycosyl‐substituted dicarboxylate detergents (DCODs) in which we optimized the polar head to clamp the membrane domain by including, on one side, two carboxyl groups that form salt bridges with basic residues abundant at the membrane–cytoplasm interface of MPs and, on the other side, a sugar to form hydrogen bonds. Upon extraction, the DCODs 8 b, 8 c, and 9 b preserved the ATPase function of BmrA, an ATP‐binding cassette pump, much more efficiently than reference or recently designed detergents. The DCODs 8 a, 8 b, 8 f, 9 a, and 9 b induced thermal shifts of 20 to 29 °C for BmrA and of 13 to 21 °C for the native version of the G‐protein‐coupled adenosine receptor A2AR. Compounds 8 f and 8 g improved the diffraction resolution of BmrA crystals from 6 to 4 Å. DCODs are therefore considered to be promising and powerful tools for the structural biology of MPs. [ABSTRACT FROM AUTHOR]

In their Research Article (e202302003), Daniel Huster et al. analyze the dynamics of the human growth hormone secretagogue receptor. The study reveals insights into the energy landscape of the molecule. Keywords: Detektoranalyse; GPCR; Membranproteine; Molekulare Dynamik; NMR-Spektroskopie DE Detektoranalyse GPCR Membranproteine Molekulare Dynamik NMR-Spektroskopie 1 1 1 08/23/23 20230828 NES 230828 B Membrane Proteins b . [Extracted from the article]

SpyTag is a peptide that forms a spontaneous amide bond with its protein partner SpyCatcher. This protein superglue is a broadly useful tool for molecular assembly, locking together biological building blocks efficiently and irreversibly in diverse architectures. We initially developed SpyTag and SpyCatcher by rational design, through splitting a domain from a Gram-positive bacterial adhesin. In this work, we established a phage-display platform to select for specific amidation, leading to an order of magnitude acceleration for interaction of the SpyTag002 variant with the SpyCatcher002 variant. We show that the 002 pair bonds rapidly under a wide range of conditions and at either protein terminus. SpyCatcher002 was fused to an intimin derived from enterohemorrhagic Escherichia coli. SpyTag002 reaction enabled specific and covalent decoration of intimin for live cell fluorescent imaging of the dynamics of the bacterial outer membrane as cells divide. [ABSTRACT FROM AUTHOR]

The interplay between membrane proteins and the lipids of the membrane is important for cellular function, however, tools enabling the interrogation of protein dynamics within native lipid environments are scarce and often invasive. We show that the styrene-maleic acid lipid particle (SMALP) technology can be coupled with hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate membrane protein conformational dynamics within native lipid bilayers. We demonstrate changes in accessibility and dynamics of the rhomboid protease GlpG, captured within three different native lipid compositions, and identify protein regions sensitive to changes in the native lipid environment. Our results illuminate the value of this approach for distinguishing the putative role(s) of the native lipid composition in modulating membrane protein conformational dynamics. [ABSTRACT FROM AUTHOR]

Described is a novel, laser-initiated radical trifluoromethylation for protein footprinting and its broad residue coverage. .CF3 reacts with 18 of the 20 common amino acids, including Gly, Ala, Ser, Thr, Asp, and Glu, which are relatively silent with regard to .OH. This new approach to footprinting is a bridge between trifluoromethylation in materials and medicinal chemistry and structural biology and biotechnology. Its application to a membrane protein and to myoglobin show that the approach is sensitive to protein conformational change and solvent accessibility. [ABSTRACT FROM AUTHOR]

Vibrational energy transfer (VET) of proteins at cell membrane plays critical roles in controlling the protein functionalities, but its detection is very challenging. By using a surface-sensitive femtosecond time-resolved sum-frequency generation vibrational spectroscopy with infrared pump, the detection of the ultrafast VET in proteins at cell membrane has finally become possible. The vibrational relaxation time of the N−H groups is determined to be 1.70(±0.05) ps for the α-helix located in the hydrophobic core of the lipid bilayer and 0.9(±0.05) ps for the membrane-bound β-sheet structure. The N−H groups with strong hydrogen bonding gain faster relaxation time. By pumping the amide A band and probing amide I band, the vibrational relaxation from N−H mode to C=O mode through two pathways (direct coupling and through intermediate states) is revealed. The ratio of the pathways depends on the NH⋅⋅⋅O=C hydrogen-bonding strength. Strong hydrogen bonding favors the coupling through intermediate states. [ABSTRACT FROM AUTHOR]

Interferon-induced transmembrane protein 3 (IFITM3) is an antiviral transmembrane protein that is thought to serve as the primary factor for inhibiting the replication of a large number of viruses, including West Nile virus, Dengue virus, Ebola virus, and Zika virus. Production of this 14.5 kDa, 133-residue transmembrane protein, especially with essential posttranslational modifications, by recombinant expression is challenging. In this report, we document the chemical synthesis of IFTIM3 in multi-milligram quantities (>15 mg) and the preparation of phosphorylated and fluorescent variants. The synthesis was accomplished by using KAHA ligations, which operate under acidic aqueous/organic mixtures that excel at solubilizing even the exceptionally hydrophobic C-terminal region of IFITM3. The synthetic material is readily incorporated into model vesicles and forms the basis for using synthetic, homogenous IFITM3 and its derivatives for further studying its structure and biological mode of action. [ABSTRACT FROM AUTHOR]

Polymer-based nanodiscs are valuable tools in biomedical research that can offer a detergent-free solubilization of membrane proteins maintaining their native lipid environment. Herein, we introduce a novel ca. 1.6 kDa SMA-based polymer with styrene:maleic acid moieties that can form nanodiscs containing a planar lipid bilayer which are useful to reconstitute membrane proteins for structural and functional studies. The physicochemical properties and the mechanism of formation of polymer-based nanodiscs are characterized by light scattering, NMR, FT-IR, and TEM. A remarkable feature is that nanodiscs of different sizes, from nanometer to sub-micrometer diameter, can be produced by varying the lipid-to-polymer ratio. The small-size nanodiscs (up to ca. 30 nm diameter) can be used for solution NMR spectroscopy studies whereas the magnetic-alignment of macro-nanodiscs (diameter of > ca. 40 nm) can be exploited for solid-state NMR studies on membrane proteins. [ABSTRACT FROM AUTHOR]

G-protein-coupled-receptors (GPCRs) are of fundamental importance for signal transduction through cell membranes. This makes them important drug targets, but structure-based drug design (SBDD) is still hampered by the limitations for structure determination of unmodified GPCRs. We show that the interligand NOEs for pharmacophore mapping (INPHARMA) method can provide valuable information on ligand poses inside the binding site of the unmodified human A2A adenosine receptor reconstituted in nanodiscs. By comparing experimental INPHARMA spectra with back-calculated spectra based on ligand poses obtained from molecular dynamics simulations, a complex structure for A2AR with the low-affinity ligand 3-pyrrolidin-1-ylquinoxalin-2-amine was determined based on the X-ray structure of ligand ZM-241,358 in complex with a modified A2AR. [ABSTRACT FROM AUTHOR]

Paramagnetische Metallionen liefern strukturelle Informationen, sowohl in EPR ‐ als auch in Festkörper ‐ NMR ‐ Experimenten, und ermöglichen einen vielversprechenden synergetischen Ansatz zum Studium von Biomakromolekülen. Wir demonstrieren die spektralen Konsequenzen einer Mg2+/Mn2+ ‐ Substitution und den daraus resultierenden Informationsgehalt für zwei verschiedene ATP:Mg2+ ‐ angetriebene Proteinmaschinen, einer im bakteriellen Replisom aktiven DnaB ‐ Helikase von Helicobacter pylori und dem ABC ‐ Transporter BmrA, einer bakteriellen Effluxpumpe. EPR ‐ Spektren belegen die Metallbindung und liefern Informationen über die Geometrie der Metallzentren in den Proteinen, und die in den NMR ‐ Spektren identifizierten paramagnetischen Relaxationsbeschleunigungen ermöglichen die Lokalisierung von Resten an der Bindungsstelle. Proteinmaschinen sind ubiquitär, und die hier beschriebenen Methoden sollten in einem breiten Kontext angewendet werden können. Spektren ändern sich als Folge einer Mg2+/Mn2+ ‐ Substitution in zwei verschiedenen ATP:Mg2+ ‐ angetriebenen Proteinmaschinen: einer DnaB ‐ Helikase und dem ABC ‐ Transporter BmrA. Während EPR ‐ Studien das Binden des Metalls bestätigen und Informationen über die Geometrie der Metallzentren liefern, identifizieren paramagnetische Relaxationsbeschleunigungen in NMR ‐ Studien die Reste an der Bindungsstelle. [ABSTRACT FROM AUTHOR]

Struktur, Dynamik und Funktion von Membranproteinen sind eng verknüpft mit den Eigenschaften der Membranumgebung, in die sie eingebettet sind. Im Allgemeinen müssen Membranproteine für strukturelle und biophysikalische Untersuchungen aber aus der Membran extrahiert und in einem geeigneten Membranmimetikum rekonstituiert werden. Ob die funktionelle und strukturelle Integrität in solchen künstlichen Umgebungen erhalten bleibt, ist oft nicht leicht zu evaluieren, aber für die Interpretation von höchster Wichtigkeit. Das Styrol ‐ Maleinsäure ‐ Copolymer ermöglicht es, Membranproteine mit den umgebenden Lipiden direkt aus nativen Membranen zu extrahieren. Die resultierenden scheibenförmigen Proteolipid ‐ Partikel werden auch native Nanoscheiben genannt. Wir zeigen hier, am Beispiel des 2×34 kDa großen bakteriellen Kationendiffusionsunterstützers CzcD, dass hochaufgelöste Festkörper ‐ NMR ‐ Spektren von integralen Membranproteinen in nativen Nanoscheiben erhalten werden können. Hochaufgelöste Festkörper ‐ NMR ‐ Spektren können für ein Membranprotein erhalten werden, das direkt mittels Styrol ‐ Maleinsäure ‐ Copolymer aus der Membran extrahiert wurde und native Nanoscheiben bildet. [ABSTRACT FROM AUTHOR]

Small-residue-mediated interhelical packings are ubiquitously found in helical membrane proteins, although their interaction dynamics and lipid dependence remain mostly uncharacterized. We used a single-pair FRET technique to examine the effect of a GXXXG motif on the association of de novo designed (AALALAA)3 helices in liposomes. Dimerization occurred with sub-second lifetimes, which was abolished by cholesterol. Utilizing the nearly instantaneous time-resolution of 2D IR spectroscopy, parallel and antiparallel helix associations were identified by vibrational couplings across helices at their interface. Taken together, the data illustrate that the GXXXG motif controls helix packing but still allows for a dynamic and lipid-regulated oligomeric state. [ABSTRACT FROM AUTHOR]

The active transport of ions across biological membranes requires their hydration shell to interact with the interior of membrane proteins. However, the influence of the external lipid phase on internal dielectric dynamics is hard to access by experiment. Using the octahelical transmembrane architecture of the copper-transporting P1B-type ATPase from Legionella pneumophila as a model structure, we have established the site-specific labeling of internal cysteines with a polarity-sensitive fluorophore. This enabled dipolar relaxation studies in a solubilized form of the protein and in its lipid-embedded state in nanodiscs. Time-dependent fluorescence shifts revealed the site-specific hydration and dipole mobility around the conserved ion-binding motif. The spatial distribution of both features is shaped significantly and independently of each other by membrane lateral pressure. [ABSTRACT FROM AUTHOR]

Für strukturelle und dynamische Untersuchungen von Membranproteinen werden heutzutage Tenside eingesetzt, in denen die strukturelle Integrität der Membranproteine meistens erhalten bleibt, ihre Funktionalität jedoch oft eingeschränkt wird. Diese Funktionalität kann in den meisten Fällen in einer Lipidumgebung wiederhergestellt werden. Hier zeigen wir mit Aminosäuren-spezifischer Auflösung, dass OmpX eine stabile β-Fass-3D-Struktur sowohl in DPC-Tensidmizellen als auch in DHPC/DMPC-Bizellen und DMPC-Nanoscheiben bilden kann. Die Dynamiken dagegen unterscheiden sich stark im Piko/Nanosekunden- und im Mikro/Millisekundenbereich zwischen der Tensid- und Lipidumgebung. Vor allem in den β-Strängen konnte eine hohe dynamische Variabilität für die Lipidumgebung festgestellt werden, die in der Tensidumgebung nicht vorhanden war. Dieses membranabhängige Verhalten könnte einen Zusammenhang für den häufig auftretenden Aktivitätsverlust von Membranproteinen in Gegenwart von Tensiden aufzeigen. [ABSTRACT FROM AUTHOR]

The chemokine receptor CXCR3 is a G protein-coupled receptor that conveys extracellular signals into cells by changing its conformation upon ligand binding. We previously hypothesized that small-molecule allosteric CXCR3-agonists do not bind to the same allosteric binding pocket as 8-azaquinazolinone-based negative allosteric modulators. We have now performed molecular-dynamics (MD) simulations with metadynamics enhanced sampling on the CXCR3 system to refine structures and binding modes and to predict the CXCR3-binding affinities of the biased allosteric agonist FAUC1036 and the negative allosteric modulator RAMX3. We have identified two distinct binding sites; a 'shallow' and a second 'deeper' pocket to which the biased allosteric agonist FAUC1036 and negative allosteric modulator RAMX3 bind, respectively. [ABSTRACT FROM AUTHOR]

Cytochrome (cyt) c transports electrons from Complex III to Complex IV in mitochondria. Cyt c is ordinarily anchored to the mitochondrial membrane through interaction with cardiolipin (CL), however its release into the cytosol initiates apoptosis. The cyt c interaction site with CL-containing bicelles was characterized by NMR spectroscopy. Chemical shift perturbations in cyt c signals upon interaction with bicelles revealed that a relatively wide region, which includes the A-site, the CXXCH motif, and the N- and C-terminal helices, and contains multiple Lys residues, interacts cooperatively with CL. The specific cyt c-CL interaction increased with increasing CL molecules in the bicelles. The location of the cyt c interaction site for CL was similar to those for Complex III and Complex IV, thus indicating that cyt c recognizes lipids and partner proteins in a similar way. In addition to elucidating the cyt c membrane-binding site, these results provide insight into the dynamic aspect of cyt c interactions in mitochondria. [ABSTRACT FROM AUTHOR]

The Min proteins from E.coli position the bacterial cell-division machinery through pole-to-pole oscillations. In vitro, Min protein self-organization can be reconstituted in the presence of a lipid membrane as a catalytic surface. However, Min dynamics have so far not been reconstituted in fully membrane-enclosed volumes. Microdroplets interfaced by lipid monolayers were employed as a simple 3D mimic of cellular compartments to reconstitute Min protein oscillations. We demonstrate that lipid monolayers are sufficient to fulfil the catalytic role of the membrane and thus represent a facile platform to investigate Min protein regulated dynamics of the cell-division protein FtsZ-mts. In particular, we show that droplet containers reveal distinct Min oscillation modes, and reveal a dependence of FtsZ-mts structures on compartment size. Finally, co-reconstitution of Min proteins and FtsZ-mts in droplets yields antagonistic localization, thus demonstrating that droplets indeed support the analysis of complex bacterial self-organization in confined volumes. [ABSTRACT FROM AUTHOR]

1H‐Detektion kann die spektrale Empfindlichkeit in der Festkörper‐NMR‐Spektroskopie stark verbessern und erlaubt dadurch Studien komplexerer Proteine. Zur 1H‐Detektion müssen jedoch austauschbare Wasserprotonen in ansonsten perdeuterierte Proteine eingeführt werden. Diese Vorbedingung hat bisweilen Studien wasserunzugänglicher Proteine stark erschwert. Hier präsentieren wir eine Methode, welche die hochaufgelöste 1H‐Detektion wasserunzugänglicher Proteine ermöglicht und selbst in hochkomplexen Medien wie zellulären Oberflächen funktioniert. Wir demonstrieren unsere Methode am Beispiel des K+‐Kanals KcsA in Liposomen sowie in situ in nativen bakteriellen Zellmembranen. Wir verwenden unsere Daten für eine Analyse der KcsA‐Dynamik und zeigen, dass der Selektivitätsfilter, der in K+‐Kanälen hochkonserviert vorliegt und kritisch für die Ionenleitfähigkeit ist, ausgeprägte molekulare Flexibilität aufweist. Im Inneren der Membran: Eine Methode wird vorgestellt, die hochaufgelöste 1H‐Festkörper‐NMR‐Spektren von wasserunzugänglichen Proteinen direkt in nativen Zellmembranen liefert. Die neue Technik wurde genutzt, um die Dynamik eines Ionenkanals in vitro und in situ zu erforschen. [ABSTRACT FROM AUTHOR]

Dengue virus nonstructural protein 4B (NS4B) is a membrane protein consisting of 248 residues with a crucial role in virus replication and interference with the host innate immunity. The dengue virus serotype 3 NS4B was reconstituted into lyso-myristoyl phosphatidylglycerol (LMPG) micelles. Backbone resonance assignment of NS4B was obtained using conventional solution NMR experiments. Further studies suggested that NS4B contained eleven helices and six of them form five potential transmembrane regions. This study provides atomic level information for an important drug target to control flavivirus infections. [ABSTRACT FROM AUTHOR]

The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero-oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro-electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter-subunit interaction and the intra-subunit conformational changes in hetero-oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein-ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment. [ABSTRACT FROM AUTHOR]

15N‐Spinrelaxationsraten können wichtige Informationen über Fernstruktur und interne Bewegungen von Membranproteinen bereitstellen. Kombiniert mit einem verbesserten Berechnungsprotokoll offenbart die mittels Relaxationsraten abgeleitete Struktur des 283 Reste umfassenden humanen spannungsabhängigen Anionenkanals ein anisotrop geformtes Fass mit einer fest verankerten N‐terminalen Helix. Diese Arbeit eröffnet somit einen NMR‐spektroskopischen Ansatz, um die Struktur und Dynamik von Membranproteinen der Säugetiere mit hoher Exaktheit und Auflösung zu bestimmen. Entspann dich! Mithilfe von 15N‐Spinrelaxation lässt sich die dreidimensionale Struktur komplexer Membranproteine ermitteln. Die von Relaxationsraten abgeleitete Struktur des humanen spannungsabhängigen Anionenkanals zeigt einzigartige Merkmale eines anisotrop geformten Fasses mit einer fest verankerten N‐terminalen Helix. [ABSTRACT FROM AUTHOR]

For applications in synthetic biology, for example, the bottom-up assembly of biomolecular nanofactories, modules of specific and controllable functionalities are essential. Of fundamental importance in such systems are energizing modules, which are able to establish an electrochemical gradient across a vesicular membrane as an energy source for powering other modules. Light-driven proton pumps like proteorhodopsin (PR) are excellent candidates for efficient energy conversion. We have extended the versatility of PR by implementing an on/off switch based on reversible chemical modification of a site-specifically introduced cysteine residue. The position of this cysteine residue in PR was identified by structure-based cysteine mutagenesis combined with a proton-pumping assay using E. coli cells overexpressing PR and PR proteoliposomes. The identified PR mutant represents the first light-driven proton pump that can be chemically switched on/off depending on the requirements of the molecular system. [ABSTRACT FROM AUTHOR]

ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1F0-ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode-assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products. [ABSTRACT FROM AUTHOR]

β-barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three-dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β-strands. Here, we employ hydrogen-deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue-specific kinetics of interstrand hydrogen-bond formation were found to be uniform in the entire β-barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long-lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate-limiting transition state and thus appears cooperative on the overall folding time scale. [ABSTRACT FROM AUTHOR]

The membrane-proximal external region (MPER) of HIV gp41 is an established target of antibodies that neutralize a broad range of HIV isolates. To evaluate the role of the transmembrane (TM) domain, synthetic MPER-derived peptides were incorporated into lipid nanoparticles using natural and designed TM domains, and antibody affinity was measured using immobilized and solution-based techniques. Peptides incorporating the native HIV TM domain exhibit significantly stronger interactions with neutralizing antibodies than peptides with a monomeric TM domain. Furthermore, a peptide with a trimeric, three-helix bundle TM domain recapitulates the binding profile of the native sequence. These studies suggest that neutralizing antibodies can bind the MPER when the TM domain is a three-helix bundle and this presentation could influence the binding of neutralizing antibodies to the virus. Lipid-bilayer presentation of viral antigens in Nanodiscs is a new platform for evaluating neutralizing antibodies. [ABSTRACT FROM AUTHOR]

Interactions between membrane proteins and lipids are often crucial for structure and function yet difficult to define because of their dynamic and heterogeneous nature. Here, we use mass spectrometry to demonstrate that membrane protein oligomers ejected from nanodiscs in the gas phase retain large numbers of lipid interactions. The complex mass spectra that result from gas-phase dissociation were assigned using a Bayesian deconvolution algorithm together with mass defect analysis, allowing us to count individual lipid molecules bound to membrane proteins. Comparison of the lipid distributions measured by mass spectrometry with molecular dynamics simulations reveals that the distributions correspond to distinct lipid shells that vary according to the type of protein-lipid interactions. Our results demonstrate that nanodiscs offer the potential for native mass spectrometry to probe interactions between membrane proteins and the wider lipid environment. [ABSTRACT FROM AUTHOR]

1H-detection can greatly improve spectral sensitivity in biological solid-state NMR (ssNMR), thus allowing the study of larger and more complex proteins. However, the general requirement to perdeuterate proteins critically curtails the potential of 1H-detection by the loss of aliphatic side-chain protons, which are important probes for protein structure and function. Introduced herein is a labelling scheme for 1H-detected ssNMR, and it gives high quality spectra for both side-chain and backbone protons, and allows quantitative assignments and aids in probing interresidual contacts. Excellent 1H resolution in membrane proteins is obtained, the topology and dynamics of an ion channel were studied. This labelling scheme will open new avenues for the study of challenging proteins by ssNMR. [ABSTRACT FROM AUTHOR]

SNARE proteins are the core machinery to drive fusion of a vesicle with its target membrane. Inspired by the tethering proteins that bridge the membranes and thus prepare SNAREs for docking and fusion, we developed a lipid-conjugated ssDNA mimic that is capable of regulating SNARE function, in situ. The DNA–lipid tethers consist of a 21 base pairs binding segment at the membrane distal end that can bridge two liposomes via specific base-pair hybridization. A linker at the membrane proximal end is used to control the separation distance between the liposomes. In the presence of these artificial tethers, SNARE-mediated lipid mixing is significantly accelerated, and the maximum fusion rate is obtained with the linker shorter than 40 nucleotides. As a programmable tool orthogonal to any native proteins, the DNA–lipid tethers can be further applied to regulate other biological processes where capturing and bridging of two membranes are the prerequisites for the subsequent protein function. [ABSTRACT FROM AUTHOR]

MAS ‐ NMR ‐ Spektroskopie wurde verwendet, um die Struktur und Dynamik der Membrananker ‐ Domäne von YadA (YadA ‐ M) direkt in der bakteriellen Außenmembran zu untersuchen, in der das Protein exprimiert wurde. YadA ist ein Adhäsin des Pathogens Yersinia enterocolitica, das an Interaktionen mit Wirtszellen beteiligt ist und darüber hinaus auch als Modellprotein zu Studien des Autotransportprozesses dient. Mithilfe von 13C ‐ 13C ‐ DARR ‐ und PDSD ‐ Spektren bei unterschiedlichen Mischzeiten konnten wir existierende chemische Verschiebungen für einen weiten Bereich von YadA ‐ M in der Lipidmembran übertragen. Die chemischen Verschiebungen von YadA ‐ M waren in den meisten Regionen im Vergleich zu denen in mikrokristallinen Präparationen, auf deren Basis bereits eine Struktur gelöst wurde, unverändert. Das trifft insbesondere auf die ASSA ‐ Region zu, die vermutlich als Übergangszustand während des Transports der löslichen Domäne eine Haarnadelstruktur einnimmt. Vergleiche der Dynamik zwischen mikrokristallinen und in der Membran eingebetteten Proben zeigen eine höhere Flexibilität der ASSA ‐ Region in den Außenmembran ‐ Präparationen bei physiologischen Temperaturen. Diese Studie wird den Weg für die MAS ‐ NMR ‐ basierte Strukturberechnung von Membranproteinen und für ein besseres Verständnis der Funktion von dynamischen Proteinbereichen in nativen Membranen bereiten. In nativer Umgebung: Festkörper ‐ NMR ‐ Spektroskopie wurde genutzt, um die Struktur und Dynamik der Membrananker ‐ Domäne von YadA direkt in der Außenmembran von E. coli zu untersuchen, in der das Protein exprimiert wurde. YadA ist ein Adhäsin des Pathogens Yersinia enterocolitica und beteiligt an Interaktionen mit der Wirtszelle; es ist auch ein Modellprotein für Studien zum Autotransportprozess. [ABSTRACT FROM AUTHOR]

Transmembrane proteins are critical for signaling, transport, and metabolism, yet their reconstitution in synthetic membranes is often challenging. Non-enzymatic and chemoselective methods to generate phospholipid membranes in situ would be powerful tools for the incorporation of membrane proteins. Herein, the spontaneous reconstitution of functional integral membrane proteins during the de novo synthesis of biomimetic phospholipid bilayers is described. The approach takes advantage of bioorthogonal coupling reactions to generate proteoliposomes from micelle-solubilized proteins. This method was successfully used to reconstitute three different transmembrane proteins into synthetic membranes. This is the first example of the use of non-enzymatic chemical synthesis of phospholipids to prepare proteoliposomes. [ABSTRACT FROM AUTHOR]

Membrane proteins may be influenced by the environment, and they may be unstable in detergents or fail to crystallize. As a result, approaches to characterize structures in a native environment are highly desirable. Here, we report a novel general strategy for precise distance measurements on outer membrane proteins in whole Escherichia coli cells and isolated outer membranes. The cobalamin transporter BtuB was overexpressed and spin-labeled in whole cells and outer membranes and interspin distances were measured to a spin-labeled cobalamin using pulse EPR spectroscopy. A comparative analysis of the data reveals a similar interspin distance between whole cells, outer membranes, and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein-protein/ligand interactions at surface-exposed sites of membrane protein complexes in whole cells and native membranes, and provides a method to validate outer membrane protein structures in their native environment. [ABSTRACT FROM AUTHOR]

Using a combination of metabolically labeled glycans, a bioorthogonal copper(I)-catalyzed azide-alkyne cycloaddition, and the controlled bleaching of fluorescent probes conjugated to azide- or alkyne-tagged glycans, a sufficiently low spatial density of dye-labeled glycans was achieved, enabling dynamic single-molecule tracking and super-resolution imaging of N-linked sialic acids and O-linked N-acetyl galactosamine (GalNAc) on the membrane of live cells. Analysis of the trajectories of these dye-labeled glycans in mammary cancer cells revealed constrained diffusion of both N- and O-linked glycans, which was interpreted as reflecting the mobility of the glycan rather than to be caused by transient immobilization owing to spatial inhomogeneities on the plasma membrane. Stochastic optical reconstruction microscopy (STORM) imaging revealed the structure of dynamic membrane nanotubes. [ABSTRACT FROM AUTHOR]

Lipidic cubic phases (LCPs) are used in areas ranging from membrane biology to biodevices. Because some membrane proteins are notoriously unstable at room temperature, and available LCPs undergo transformation to lamellar phases at low temperatures, development of stable low-temperature LCPs for biophysical studies of membrane proteins is called for. Monodihydrosterculin (MDS) is a designer lipid based on monoolein (MO) with a configurationally restricted cyclopropyl ring replacing the olefin. Small-angle X-ray scattering (SAXS) analyses revealed a phase diagram for MDS lacking the high-temperature, highly curved reverse hexagonal phase typical for MO, and extending the cubic phase boundary to lower temperature, thereby establishing the relationship between lipid molecular structure and mesophase behavior. The use of MDS as a new material for LCP-based membrane protein crystallization at low temperature was demonstrated by crystallizing bacteriorhodopsin at 20 °C as well as 4 °C. [ABSTRACT FROM AUTHOR]

Die Struktur von Proteinen steht in engem Zusammenhang mit ihrer Funktion, wobei gefaltete Proteinstrukturen Erkennungsmotive zur Bindung von Liganden und anderen Proteinen darstellen. Die Massenspektrometrie (MS) kann Informationen auf verschiedenen Ebenen der Proteinstruktur zur Verfügung stellen, angefangen bei der primären Aminosäuresequenz bis hin zur dreidimensionalen Faltung und Wechselwirkungen innerhalb der Quartärstruktur. Da es sich bei der MS um ein Gasphasenverfahren handelt, scheint es vielleicht widersinnig, sie zur Untersuchung der Struktur und nichtkovalenten Wechselwirkungen von Proteinen in Lösung zu nutzen. Wir zeigen hier jedoch durch neue Einblicke in dynamische Netzwerke aus Wechselwirkungen, Dissoziationsmechanismen und die Kooperativität der Ligandenbindung, dass MS ‐ Untersuchungen über die simple Erhaltung von Proteinwechselwirkungen in der Gasphase hinausgehen können. Wir ziehen mögliche Stolpersteine bei der Dateninterpretation in Betracht und legen einen besonderen Schwerpunkt auf jüngste Untersuchungen, die quantitative Informationen über dynamische Proteinwechselwirkungen sowohl in löslichen als auch in Membran ‐ gebundenen Proteinanordnungen liefern. Infos massenweise: Die Massenspektrometrie kann Informationen über dynamische Netzwerke aus Wechselwirkungen, Dissoziationsmechanismen und die Kooperativität der Ligandenbindung in Lösung liefern. Dieser Aufsatz erörtert mögliche Fehlerquellen bei der Dateninterpretation, mit einem Schwerpunkt auf aktuellen Untersuchungen, in denen quantitative Informationen über dynamische Proteinwechselwirkungen in löslichen wie auch in Membran ‐ eingebetteten Proteinanordnungen erhalten wurden. [ABSTRACT FROM AUTHOR]

G-protein-coupled receptors (GPCRs) exist in conformational equilibrium between active and inactive states, and the former population determines the efficacy of signaling. However, the conformational equilibrium of GPCRs in lipid bilayers is unknown owing to the low sensitivities of their NMR signals. To increase the signal intensities, a deuteration method was developed for GPCRs expressed in an insect cell/baculovirus expression system. The NMR sensitivities of the methionine methyl resonances from the β2-adrenergic receptor (β2AR) in lipid bilayers of reconstituted high-density lipoprotein (rHDL) increased by approximately 5-fold upon deuteration. NMR analyses revealed that the exchange rates for the conformational equilibrium of β2AR in rHDLs were remarkably different from those measured in detergents. The timescales of GPCR signaling, calculated from the exchange rates, are faster than those of receptor tyrosine kinases and thus enable rapid neurotransmission and sensory perception. [ABSTRACT FROM AUTHOR]

Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opportunity to compare the properties of an integral membrane protein in different artificial lipid/surfactant environments with those in the native bilayer. Here, we demonstrate that reaction centers purified using a styrene maleic acid copolymer remain associated with a complement of native lipids and do not display the modified functional properties that typically result from detergent solubilization. Direct comparisons show that reaction centers are more stable in this copolymer/lipid environment than in a detergent micelle or even in the native membrane, suggesting a promising new route to exploitation of such photovoltaic integral membrane proteins in device applications. [ABSTRACT FROM AUTHOR]

Eine neue Methode zur chemischen Proteinmarkierung läuft mit hoher Zielspezifität innerhalb weniger Minuten ab und ermöglicht eine freie Wahl des Reportermoleküls. Sie beruht auf Peptidtemplaten, die einen Thioester und ein N-terminales Cystein so ausrichten, dass eine Acyltransferreaktion bei nanomolarer Konzentration möglich wird. Das Zielprotein wird N-terminal mit einem 22 Aminosäure langen Cys-E3-Peptid (Akzeptor) ausgestattet, das mit einem Reporter-tragenden K3-Peptid (Donor) ein Coiled-Coil bilden kann. Dadurch wird die Übertragung des Reportermoleküls auf den Akzeptor am Zielprotein ausgelöst. Weil die Ligierung der wechselwirkenden Peptide vermieden wird, ist der Massenzuwachs am Zielprotein minimal. Zur Veranschaulichung der Methode werden die rasche Fluoreszenzmarkierung und die fluoreszenzmikroskopische Untersuchung des humanen Y2-Rezeptors an lebenden Zellen gezeigt. [ABSTRACT FROM AUTHOR]

Difficulties in constructing complex lipid/protein membranes have severely limited the development of functional artificial cells endowed with vital membrane-related functions. The Sec translocon membrane channel, which mediates the insertion of membrane proteins into the plasma membrane, was constructed in the membrane of lipid vesicles through in vitro expression of its component proteins. The components of the Sec translocon were synthesized from their respective genes in the presence of liposomes, thereby bringing about a functional complex. The synthesized E. coli Sec translocon mediated the membrane translocation of single- and multi-span membrane proteins. The successful translocation of a functional peptidase into the liposome lumen further confirmed the proper insertion of the translocon complex. Our results demonstrate the feasible construction of artificial cells, the membranes of which can be functionalized by directly decoding genetic information into membrane functions. [ABSTRACT FROM AUTHOR]

We propose a novel single-cell chemical proteomics (SCCP) strategy to profile low-abundance membrane proteins in single cells. In this approach, the membrane protein GB1 and its splicing variants were targeted on cultured cell lines and primary neurons using a specifically designed activity-based probe. The functionally labeled single cells were encapsulated in individual buffer droplets on a PDMS microwell array, and were further picked up one at a time and loaded into a capillary electrophoresis system for cell lysis, separation, and laser-induced fluorescence detection of the targeted proteins. The results revealed the expression of GB1 splicing variants in HEK and MEF cells, which was previously only suggested at the transcriptional level. We further applied this method to investigate single primary cells and observed significant heterogeneity among individual mouse cerebellar granule neurons. Interference experiments with GB1 antagonist and agonist validated this observation. [ABSTRACT FROM AUTHOR]

Solid-state NMR is a powerful tool for studying membrane proteins in a native-like lipid environment. 3D magic angle spinning (MAS) NMR was employed to characterize the structure of E.coli diacylglycerol kinase (DAGK) reconstituted into its native E.coli lipid membranes. The secondary structure and topology of DAGK revealed by solid-state NMR are different from those determined by solution-state NMR and X-ray crystallography. This study provides a good example for demonstrating the influence of membrane environments on the structure of membrane proteins. [ABSTRACT FROM AUTHOR]

NMR spectroscopy is used to detect site-specific intermolecular short-range contacts in a membrane-protein-chaperone complex. This is achieved by an 'orthogonal' isotope-labeling scheme that permits the unambiguous detection of intermolecular NOEs between the well-folded chaperone and the unfolded substrate ensemble. The residues involved in these contacts are part of the chaperone-substrate contact interface. The approach is demonstrated for the 70 kDa bacterial Skp-tOmpA complex. [ABSTRACT FROM AUTHOR]

Die Effektivität von Wirkstoffen und Biomolekülen basiert auf ihrer Fähigkeit, die Lipidmembran zu passieren. Die Entwicklung von Methoden, diese Transportprozesse direkt und mit hoher Empfindlichkeit zu verfolgen, bleibt eine Herausforderung. Der Einschluss eines chemosensorischen Ensembles, bestehend aus einem makrocyclischen Wirt (p ‐ Sulfonatocalix[4]aren oder Cucurbit[7]uril) und einem Fluoreszenzfarbstoff (Lucigenin oder Berberin) in Liposomen ermöglicht es, den Membrandurchtritt von unmarkierten bioorganischen Molekülen direkt und in Echtzeit mittels Fluoreszenz zu verfolgen. Dieser In ‐ vitro ‐ Assay kann auf unterschiedliche Kanalproteine und Analyten angewendet werden, eignet sich zur schnellen Charakterisierung von Kanalmodulatoren und liefert zudem die absolute Kinetik der Translokation. Mit dieser neuen biophysikalischen Methode konnten wir erstmalig die direkte schnelle Translokation des antibiotisch wirksamen Peptids Protamin durch das bakterielle Transmembranprotein OmpF nachweisen. Eine direkte Echtzeitdetektion des Analyttransports durch eine Lipiddoppelschicht ‐ Membran gelingt durch den Einschluss eines Makrocyclus und eines Fluoreszenzfarbstoffs in Liposomen. Sobald der Analyt die Membran passiert hat, wird der Farbstoff aus dem Makrocyclus verdrängt, was mittels Fluoreszenz verfolgt werden kann. Eine Studie der Translokation eines stark argininhaltigen Peptids durch ein bakterielles Membranprotein belegt den Nutzen der Methode. [ABSTRACT FROM AUTHOR]