Biochemie und Chemie
Refine
Year of publication
Document Type
- Article (61)
- Part of a Book (1)
- Preprint (1)
Has Fulltext
- yes (63)
Is part of the Bibliography
- no (63)
Keywords
- rna (5)
- Kinases (3)
- Phosphorylation (3)
- RNA (3)
- Solution-state NMR (3)
- p63 (3)
- Biophysical chemistry (2)
- E. coli (2)
- EPR (2)
- Membrane protein (2)
- Membrane proteins (2)
- Mitophagy (2)
- Nanodisc (2)
- PELDOR (2)
- Target validation (2)
- base pairing (2)
- biophysics (2)
- quality control (2)
- structural biology (2)
- AEC syndrome (1)
- Ab-initio-Rechnungen (1)
- B. subtilis (1)
- Biochemie (1)
- Biochemistry (1)
- Biophysics and structural biology (1)
- Biopolymers in vivo (1)
- Biotin (1)
- Bradyrhizobium (1)
- Cell biology (1)
- Cell-free synthetic biology (1)
- Computational Biology (1)
- Computational models (1)
- DEER (1)
- DNA damage (1)
- Data processing (1)
- Direct carbon (1)
- Echtzeit-NMR-Spektroskopie (1)
- Eph receptors (1)
- Ephrin ligand (1)
- Esters (1)
- Eukaryota (1)
- Festkörper-DNP (1)
- Fmoc solid phase peptide synthesis (1)
- G protein-coupled receptor (GPCR) (1)
- Gemischtvalente Verbindungen (1)
- High-throughput screening (1)
- Internal ribosome entry site (1)
- Ion source (1)
- LILBID (1)
- Luciferase (1)
- Molecular Physiology of the Cell Membrane (1)
- Molecular biology (1)
- Molecular conformation (1)
- Molecular modelling (1)
- Moleküldynamik (1)
- NMR spectroscopy (1)
- Native mass spectrometry (1)
- Non-covalent mass spectrometry (1)
- Nuclear magnetic resonance (1)
- Oligomeric State (1)
- Overhauser-Effekt (1)
- PDZ domain (1)
- PELDOR/DEER (1)
- Personalisierte Medizin (1)
- Pharmacodynamics (1)
- Primary metabolites (1)
- Protein Structure and Dynamics (1)
- Protein folding (1)
- Protein translation (1)
- RNA processing (1)
- RNA recognition motif (1)
- RNA sequencing (RNA-Seq) (1)
- RNA stability (1)
- RNASolution-state NMR (1)
- RRM domain (1)
- Resonance assignment (1)
- Rpo4/7 (1)
- SAP domain (1)
- SAXS (1)
- SF3b145 (1)
- SF3b49 (1)
- Scribble (1)
- Secondary metabolites (1)
- Shine-Dalgarno (1)
- Sinorhizobium fredii (1)
- Solid-state NMR (1)
- Soluble proteins (1)
- Soybean (1)
- Ste2p (1)
- Stoffwechsel (1)
- TEMPO (1)
- Tools and resources (1)
- Topology (1)
- Translation initiation (1)
- Transporters (1)
- U2 snRNP (1)
- Wang resin (1)
- X-ray crystallography (1)
- Zellstudien (1)
- a-proteobacteria (1)
- adenine (1)
- amorphous stability (1)
- analysis (1)
- antibiotics (1)
- auto-inhibition (1)
- binding sites (1)
- biochemistry (1)
- biological chemistry (1)
- biological models (1)
- carbon and proton assignments (1)
- catabolism (1)
- cell biology (1)
- cell death (1)
- cell studies (1)
- chromatin (1)
- colorectal cancer (1)
- conformational flexibility (1)
- detection (1)
- diagnostic radiologic examination (1)
- divalent metal ions (1)
- dsRNA (1)
- dual nucleic acid binding (1)
- embryologic development (1)
- entropy (1)
- f-MLF (1)
- folding (1)
- formylation (1)
- gene expression regulation (1)
- glass forming ability (1)
- guanosine (1)
- helix (snails) (1)
- hotmelt extrusion (1)
- ions (1)
- kinetically trapped state (1)
- kinetics (1)
- ligands (1)
- magic-angle spinning (1)
- mammals (1)
- membrane protein (1)
- mesoporous silica (1)
- messenger (1)
- meta-stable structures (1)
- metabolism (1)
- metals (1)
- mice (1)
- molecular conformation (1)
- mouse (1)
- mouse model (1)
- multiple sclerosis (1)
- nESI (1)
- nuclear magnetic resonance (1)
- nuclear magnetic resonance (NMR) (1)
- nuclear magnetic resonance spectroscopy (1)
- nuclear matrix (1)
- nucleotides (1)
- oligonucleotide (1)
- oocytes (1)
- personalized medicine (1)
- phage display (1)
- phosphorylation (1)
- photolabile protection (1)
- plant symbioses (1)
- plasmid copy number (1)
- precision (1)
- protein aggregation (1)
- protein dynamics (1)
- protein folding (1)
- protein–protein interaction (1)
- protons (1)
- quorum sensing (QS) (1)
- real-time NMR spectroscopy (1)
- relapsing-remitting (1)
- ribonucleases (1)
- ribosomal binding site (1)
- ribosome (1)
- riboswitches (1)
- rna recognition motif (1)
- roentgen rays (1)
- sORF (1)
- salmonella (1)
- scaffold attachment factor proteins (1)
- secondary chemical shifts (1)
- skin (1)
- small gene (1)
- small protein (1)
- solid-state NMR (1)
- solvents (1)
- spectrum (1)
- spectrum analysis (1)
- spring-loaded activation (1)
- ssFLYA (1)
- stacking (1)
- structural biolog (1)
- sumoylation (1)
- supersaturation (1)
- synthetic riboswitches (1)
- temperature (1)
- tetracycline aptamer (1)
- thermometers (1)
- thiostrepton (1)
- titration method (1)
- transcription (1)
- transcriptional regulation (1)
- translation initiation region (1)
- tryptophan (1)
- tyrosine kinase signaling (1)
Institute
- Zentrum für Biomolekulare Magnetische Resonanz (BMRZ) (63) (remove)
The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.
The field of dynamic nuclear polarization has undergone tremendous developments and diversification since its inception more than 6 decades ago. In this review we provide an in-depth overview of the relevant topics involved in DNP-enhanced MAS NMR spectroscopy. This includes the theoretical description of DNP mechanisms as well as of the polarization transfer pathways that can lead to a uniform or selective spreading of polarization between nuclear spins. Furthermore, we cover historical and state-of-the art aspects of dedicated instrumentation, polarizing agents, and optimization techniques for efficient MAS DNP. Finally, we present an extensive overview on applications in the fields of structural biology and materials science, which underlines that MAS DNP has moved far beyond the proof-of-concept stage and has become an important tool for research in these fields.
A key function of reversible protein phosphorylation is to regulate protein–protein interactions, many of which involve short linear motifs (3–12 amino acids). Motif‐based interactions are difficult to capture because of their often low‐to‐moderate affinities. Here, we describe phosphomimetic proteomic peptide‐phage display, a powerful method for simultaneously finding motif‐based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C‐terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD‐95/Dlg/ZO‐1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR. We uncover site‐specific phospho‐regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho‐regulation of motif‐based interactions on a large scale.
The p53 family of transcription factors (p53, p63 and p73) covers a wide range of functions critical for development, homeostasis and health of mammals across their lifespan. Beside the well-established tumor suppressor role, recent evidence has highlighted novel non-oncogenic functions exerted by p73. In particular, p73 is required for multiciliated cell (MCC) differentiation; MCCs have critical roles in brain and airways to move fluids across epithelial surfaces and to transport germ cells in the reproductive tract. This novel function of p73 provides a unifying cellular mechanism for the disparate inflammatory and immunological phenotypes of p73-deficient mice. Indeed, mice with Trp73 deficiency suffer from hydrocephalus, sterility and chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance since MCCs are essential for cleaning airways from inhaled pollutants, pathogens and allergens. Cross-species genomic analyses and functional rescue experiments identify TAp73 as the master transcriptional integrator of ciliogenesis, upstream of previously known central nodes. In addition, TAp73 shows a significant ability to regulate cellular metabolism and energy production through direct transcriptional regulation of several metabolic enzymes, such as glutaminase-2 and glucose-6 phosphate dehydrogenase. This recently uncovered role of TAp73 in the regulation of cellular metabolism strongly affects oxidative balance, thus potentially influencing all the biological aspects associated with p73 function, including development, homeostasis and cancer. Although through different mechanisms, p63 isoforms also contribute to regulation of cellular metabolism, thus indicating a common route used by all family members to control cell fate. At the structural level, the complexity of p73's function is further enhanced by its ability to form heterotetramers with some p63 isoforms, thus indicating the existence of an intrafamily crosstalk that determines the global outcome of p53 family function. In this review, we have tried to summarize all the recent evidence that have emerged on the novel non-oncogenic roles of p73, in an attempt to provide a unified view of the complex function of this gene within its family.
The bile acid activated transcription factor farnesoid X receptor (FXR) regulates numerous metabolic processes and is a rising target for the treatment of hepatic and metabolic disorders. FXR agonists have revealed efficacy in treating non-alcoholic steatohepatitis (NASH), diabetes and dyslipidemia. Here we characterize imatinib as first-in-class allosteric FXR modulator and report the development of an optimized descendant that markedly promotes agonist induced FXR activation in a reporter gene assay and FXR target gene expression in HepG2 cells. Differential effects of imatinib on agonist-induced bile salt export protein and small heterodimer partner expression suggest that allosteric FXR modulation could open a new avenue to gene-selective FXR modulators.
The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.
TEMPO spin labels protected with 2-nitrobenzyloxymethyl groups were attached to the amino residues of three different nucleosides: deoxycytidine, deoxyadenosine, and adenosine. The corresponding phosphoramidites could be incorporated by unmodified standard procedures into four different self-complementary DNA and two RNA oligonucleotides. After photochemical removal of the protective group, elimination of formic aldehyde and spontaneous air oxidation, the nitroxide radicals were regenerated in high yield. The resulting spin-labeled palindromic duplexes could be directly investigated by PELDOR spectroscopy without further purification steps. Spin–spin distances measured by PELDOR correspond well to the values obtained from molecular models.
Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level.
Investigating three-dimensional (3D) structures of proteins in living cells by in-cell nuclear magnetic resonance (NMR) spectroscopy opens an avenue towards understanding the structural basis of their functions and physical properties under physiological conditions inside cells. In-cell NMR provides data at atomic resolution non-invasively, and has been used to detect protein-protein interactions, thermodynamics of protein stability, the behavior of intrinsically disordered proteins, etc. in cells. However, so far only a single de novo 3D protein structure could be determined based on data derived only from in-cell NMR. Here we introduce methods that enable in-cell NMR protein structure determination for a larger number of proteins at concentrations that approach physiological ones. The new methods comprise (1) advances in the processing of non-uniformly sampled NMR data, which reduces the measurement time for the intrinsically short-lived in-cell NMR samples, (2) automatic chemical shift assignment for obtaining an optimal resonance assignment, and (3) structure refinement with Bayesian inference, which makes it possible to calculate accurate 3D protein structures from sparse data sets of conformational restraints. As an example application we determined the structure of the B1 domain of protein G at about 250 μM concentration in living E. coli cells.
Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5′ splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.