Publications by authors named "Sascha Lange"

Polymers can facilitate detergent-free extraction of membrane proteins into nanodiscs (e.g., SMALPs, DIBMALPs), incorporating both integral membrane proteins as well as co-extracted native membrane lipids.

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Solid-state NMR allows for the study of membrane proteins under physiological conditions. Here we describe a method for detection of bound ions in the selectivity filter of ion channels using solid-state NMR. This method employs standard H-detected solid-state NMR setup and experiment types, which is enabled by using N-labelled ammonium ions to mimic potassium ions.

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Recently developed homonuclear transverse mixing optimal control pulses (hTROP) revealed an elegant way to enhance the detected signal in multidimensional magic-angle spinning (MAS) nuclear magnetic resonance experiments. Inspired by their work, we present two homonuclear simplified preservation of equivalent pathways spectroscopy (hSPEPS) sequences for recoupling CA-CO and CA-CB dipolar couplings under fast and ultrafast MAS rates, theoretically enabling a √2 improvement in sensitivity for each indirect dimension. The efficiencies of hSPEPS are evaluated for non-deuterated samples of influenza A M2 and bacterial rhomboid protease GlpG under two different external magnetic fields (600 and 1200 MHz) and MAS rates (55 and 100 kHz).

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Viroporins are small ion channels in membranes of enveloped viruses that play key roles during viral life cycles. To use viroporins as drug targets against viral infection requires in-depth mechanistic understanding and, with that, methods that enable investigations under in situ conditions. Here, we apply surface-enhanced infrared absorption (SEIRA) spectroscopy to Influenza A M2 reconstituted within a solid-supported membrane, to shed light on the mechanics of its viroporin function.

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Rhomboid proteases hydrolyze substrate helices within the lipid bilayer to release soluble domains from the membrane. Here, we investigate the mechanism of activity regulation for this unique but wide-spread protein family. In the model rhomboid GlpG, a lateral gate formed by transmembrane helices TM2 and TM5 was previously proposed to allow access of the hydrophobic substrate to the shielded hydrophilic active site.

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Protein dynamics are an intrinsically important factor when considering a protein's biological function. Understanding these motions is often limited through the use of static structure determination methods, namely, X-ray crystallography and cryo-EM. Molecular simulations have allowed for the prediction of global and local motions of proteins from these static structures.

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The flow of ions across cell membranes facilitated by ion channels is an important function for all living cells. Despite the huge amount of structural data provided by crystallography, elucidating the exact interactions between the selectivity filter atoms and bound ions is challenging. Here, we detect bound N-labeled ammonium ions as a mimic for potassium ions in ion channels using solid-state NMR under near-native conditions.

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Intramembrane proteolysis plays a fundamental role in many biological and pathological processes. Intramembrane proteases thus represent promising pharmacological targets, but few selective inhibitors have been identified. This is in contrast to their soluble counterparts, which are inhibited by many common drugs, and is in part explained by the inherent difficulty to characterize the binding of drug-like molecules to membrane proteins at atomic resolution.

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Energy-coupling factor (ECF) transporters for uptake of vitamins and transition-metal ions into prokaryotic cells share a common architecture consisting of a substrate-specific integral membrane protein (S), a transmembrane coupling protein (T) and two cytoplasmic ATP-binding-cassette-family ATPases. S components rotate within the membrane to expose their binding pockets alternately to the exterior and the cytoplasm. In contrast to vitamin transporters, metal-specific systems rely on additional proteins with essential but poorly understood functions.

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Rhomboid proteases are intramembrane proteases that hydrolyze substrate peptide bonds within the lipid bilayer and are important for a wide range of biological processes. The bacterial intramembrane protease GlpG is one of the model systems for structural investigations of the rhomboid family. Two different models of substrate gating have been proposed, based on crystal structures of GlpG in detergent micelles.

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Ion conduction through potassium channels is a fundamental process of life. On the basis of crystallographic data, it was originally proposed that potassium ions and water molecules are transported through the selectivity filter in an alternating arrangement, suggesting a "water-mediated" knock-on mechanism. Later on, this view was challenged by results from molecular dynamics simulations that revealed a "direct" knock-on mechanism where ions are in direct contact.

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Uropathogenic Escherichia coli invades and colonizes hosts by attaching to cells using adhesive pili on the bacterial surface. Although many biophysical techniques have been used to study the structure and mechanical properties of pili, many important details are still unknown. Here we use proton-detected solid-state NMR experiments to investigate solvent accessibility and structural dynamics.

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A model for hematopoiesis is presented that explicitly includes the erythrocyte, granulocyte, and thrombocyte lineages and their common precursors. A small number of stem cells proliferate and differentiate through different compartments to produce the vast number of blood cells needed every day. Growth factors regulate the proliferation of cells dependent on the current demand.

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Proton detection and fast magic-angle spinning have advanced biological solid-state NMR, allowing for the backbone assignment of complex protein assemblies with high sensitivity and resolution. However, so far no method has been proposed to detect intermolecular interfaces in these assemblies by proton detection. Herein, we introduce a concept based on methyl labeling that allows for the assignment of these moieties and for the study of protein-protein interfaces at atomic resolution.

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FGF2 is secreted from cells by an unconventional secretory pathway. This process is mediated by direct translocation across the plasma membrane. Here, we define the minimal molecular machinery required for FGF2 membrane translocation in a fully reconstituted inside-out vesicle system.

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Obtaining unambiguous resonance assignments remains a major bottleneck in solid-state NMR studies of protein structure and dynamics. Particularly for supramolecular assemblies with large subunits (>150 residues), the analysis of crowded spectral data presents a challenge, even if three-dimensional (3D) spectra are used. Here, we present a proton-detected 4D solid-state NMR assignment procedure that is tailored for large assemblies.

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Proteins are synthesized in cells by ribosomes and, in parallel, prepared for folding or targeting. While ribosomal protein synthesis is progressing, the nascent chain exposes amino-terminal signal sequences or transmembrane domains that mediate interactions with specific interaction partners, such as the signal recognition particle (SRP), the SecA-adenosine triphosphatase, or the trigger factor. These binding events can set the course for folding in the cytoplasm and translocation across or insertion into membranes.

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Binding of renin and prorenin to the (pro)renin receptor (PRR) increases their enzymatic activity and upregulates the expression of pro-fibrotic genes in vitro. Expression of PRR is increased in the heart and kidney of hypertensive and diabetic animals, but its causative role in organ damage is still unclear. To determine whether increased expression of PRR is sufficient to induce cardiac or renal injury, we generated a mouse that constitutively overexpresses PRR by knocking-in the Atp6ap2/PRR gene in the hprt locus under the control of a CMV immediate early enhancer/chicken beta-actin promoter.

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HLA-DM mediates the exchange of peptides loaded onto MHCII molecules during antigen presentation by a mechanism that remains unclear and controversial. Here, we investigated the sequence and structural determinants of HLA-DM interaction. Peptides interacting nonoptimally in the P1 pocket exhibited low MHCII binding affinity and kinetic instability and were highly susceptible to HLA-DM-mediated peptide exchange.

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Myeloperoxidase (MPO) is an enzyme expressed in neutrophils and monocytes/macrophages. Beside its well-defined role in innate immune defence, it may also be responsible for tissue damage. To identify the role of MPO in the progression of chronic kidney disease (CKD), we investigated CKD in a model of renal ablation in MPO knockout and wild-type mice.

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A feature of neurodegenerative diseases is the intraneuronal accumulation of misfolded proteins. In familial encephalopathy with neuroserpin inclusion bodies (FENIB), mutations in neuroserpin lead to accumulation of neuroserpin polymers within the endoplasmic reticulum (ER) of neurons. Cell culture based studies have shown that ER-associated degradation (ERAD) is involved in clearance of mutant neuroserpin.

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T cells participate in angiotensin II (Ang II)-induced hypertension. However, the specific subsets of T cells that are important in the end-organ damage are unknown. T-helper 17 cells are a recently identified subset that produces interleukin 17 (IL-17) and requires interleukin 23 (IL-23) for expansion.

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Membranous nephropathy (MN) accounts for most cases of the nephrotic syndrome in adults. Recently, studies on the underlying pathomechanisms led to the identification of the podocyte M-type receptor for secretory phospholipase A2 (PLA2R1) as a target antigen of circulating autoantibodies. Autoantibodies to PLA2R1 may not only play a role in the development of primary MN, but also serve as a marker for diagnosis, disease activity and therapy monitoring.

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Nuclear magnetic resonance (NMR) spectroscopy is one of the most commonly used spectroscopic techniques to obtain information on the structure and dynamics of biological and chemical materials. A variety of samples can be studied including solutions, crystalline solids, powders and hydrated protein extracts. However, biological NMR spectroscopy is limited to concentrated samples, typically in the millimolar range, due to its intrinsic low sensitivity compared to other techniques such as fluorescence or electron paramagnetic resonance (EPR) spectroscopy.

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Background And Objective: The antihypertensive effects of the direct renin inhibitor aliskiren last substantially longer after treatment withdrawal than expected based upon its plasma half-life. This may be attributable to drug accumulation in the kidney as recently shown in rats and mice. Since aliskiren binds to renin we examined in the present study whether this accumulation depends on the renin content of the kidney.

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