Probing Biomolecular Interactions with Paramagnetic Nuclear Magnetic Resonance Spectroscopy.

Recent advances in computational methods like AlphaFold have transformed structural biology, enabling accurate modeling of protein complexes and driving applications in drug discovery and protein engineering. However, predicting the structure of systems involving weak, transient, or dynamic interactions, or of complexes with disordered regions, remains challenging. Nuclear Magnetic Resonance (NMR) spectroscopy offers atomic-level insights into biomolecular complexes, even in weakly interacting and dynamic systems.

Non-canonical amino acids for site-directed spin labeling of membrane proteins.

Membrane proteins remain challenging targets for conventional structural biology techniques because they need to reside within complex hydrophobic lipid environments to maintain proper structure and function. Magnetic resonance combined with site-directed spin labeling is an alternative method that provides atomic-level structural and dynamical information from effects introduced by an electron- or nuclear-based spin label. With the advent of bioorthogonal click chemistries and genetically engineered non-canonical amino acids (ncAAs), options for linking spin probes to biomolecules have substantially broadened outside the conventional cysteine-based labeling scheme.

Structural Studies on the Binding Mode of Bisphenols to PPARγ.

Bisphenol A (BPA) and bisphenol B (BPB) are widely used in the production of plastics, and their potential adverse health effects, particularly on endocrine disruption and metabolic health, have raised concern. Peroxisome proliferator-activated receptor gamma (PPARγ) plays a pivotal role in metabolic regulation and adipogenesis, making it a target of interest in understanding the development of obesity and associated health impacts. In this study, we employ X-ray crystallography and molecular dynamics (MD) simulations to study the interaction of PPARγ with BPA and BPB.

Similar Binding Modes of cGMP Analogues Limit Selectivity in Modulating Retinal CNG Channels via the Cyclic Nucleotide-Binding Domain.

In treating , a genetic disorder causing progressive vision loss, selective inhibition of rod cyclic nucleotide-gated (CNG) channels holds promise. Blocking the increased Ca-influx in rod photoreceptors through CNG channels can potentially delay disease progression and improve the quality of life for patients. To find inhibitors for rod CNG channels, we investigated the impact of 16 cGMP analogues on both rod and cone CNG channels using the patch-clamp technique.

Targeting ion channels with ultra-large library screening for hit discovery.

Ion channels play a crucial role in a variety of physiological and pathological processes, making them attractive targets for drug development in diseases such as diabetes, epilepsy, hypertension, cancer, and chronic pain. Despite the importance of ion channels in drug discovery, the vastness of chemical space and the complexity of ion channels pose significant challenges for identifying drug candidates. The use of methods in drug discovery has dramatically reduced the time and cost of drug development and has the potential to revolutionize the field of medicine.

Ferric reduction by a CYBDOM protein counteracts increased iron availability in root meristems induced by phosphorus deficiency.

To mobilize sparingly available phosphorus (P) in the rhizosphere, many plant species secrete malate to release P sorbed onto (hydr)oxides of aluminum and iron (Fe). In the presence of Fe, malate can provoke Fe over-accumulation in the root apoplast, triggering a series of events that inhibit root growth. Here, we identified HYPERSENSITIVE TO LOW P1 (HYP1), a CYBDOM protein constituted of a DOMON and a cytochrome b561 domain, as critical to maintain cell elongation and meristem integrity under low P.

Detecting Protein-Ligand Interactions with Nitroxide Based Paramagnetic Cosolutes.

NMR spectroscopy techniques can provide important information about protein-ligand interactions. Here we tested an NMR approach which relies on the measurement of paramagnetic relaxation enhancements (PREs) arising from analogous cationic, anionic or neutral soluble nitroxide molecules, which distribute around the protein-ligand complex depending on near-surface electrostatic potentials. We applied this approach to two protein-ligand systems, interleukin-8 interacting with highly charged glycosaminoglycans and the SH2 domain of Grb2 interacting with less charged phospho-tyrosine tripeptides.

Enhancing a Sphaerobacter thermophilus ω-transaminase for kinetic resolution of β- and γ-amino acids.

Sphaerobacter thermophilus synthesizes an ω-transaminase (ω-TA) that allows the production of enantiomerically pure β-amino acids. To obtain ω-TA variants with a higher activity and more favorable properties for industrial use, we modified critical amino acid residues either in the catalytic center or in a previously proposed signature motif critical for aromatic β-amino acid ω-TAs. Seventeen different variants of this enzyme were generated and their activity was examined with four β-amino acids and one γ-amino acid, and compared with the wildtype's activity.

Recent Advances in Computer-Aided Structure-Based Drug Design on Ion Channels.

Ion channels play important roles in fundamental biological processes, such as electric signaling in cells, muscle contraction, hormone secretion, and regulation of the immune response. Targeting ion channels with drugs represents a treatment option for neurological and cardiovascular diseases, muscular degradation disorders, and pathologies related to disturbed pain sensation. While there are more than 300 different ion channels in the human organism, drugs have been developed only for some of them and currently available drugs lack selectivity.

Guiding protein design choices by per-residue energy breakdown analysis with an interactive web application.

Recent developments in machine learning have greatly facilitated the design of proteins with improved properties. However, accurately assessing the contributions of an individual or multiple amino acid mutations to overall protein stability to select the most promising mutants remains a challenge. Knowing the specific types of amino acid interactions that improve energetic stability is crucial for finding favorable combinations of mutations and deciding which mutants to test experimentally.

Recent Advances in NMR Protein Structure Prediction with ROSETTA.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful method for studying the structure and dynamics of proteins in their native state. For high-resolution NMR structure determination, the collection of a rich restraint dataset is necessary. This can be difficult to achieve for proteins with high molecular weight or a complex architecture.

Improving the Modeling of Extracellular Ligand Binding Pockets in RosettaGPCR for Conformational Selection.

G protein-coupled receptors (GPCRs) are the largest class of drug targets and undergo substantial conformational changes in response to ligand binding. Despite recent progress in GPCR structure determination, static snapshots fail to reflect the conformational space of putative binding pocket geometries to which small molecule ligands can bind. In comparative modeling of GPCRs in the absence of a ligand, often a shrinking of the orthosteric binding pocket is observed.

Structure and function of the metagenomic plastic-degrading polyester hydrolase PHL7 bound to its product.

The recently discovered metagenomic-derived polyester hydrolase PHL7 is able to efficiently degrade amorphous polyethylene terephthalate (PET) in post-consumer plastic waste. We present the cocrystal structure of this hydrolase with its hydrolysis product terephthalic acid and elucidate the influence of 17 single mutations on the PET-hydrolytic activity and thermal stability of PHL7. The substrate-binding mode of terephthalic acid is similar to that of the thermophilic polyester hydrolase LCC and deviates from the mesophilic IsPETase.

Sparse pseudocontact shift NMR data obtained from a non-canonical amino acid-linked lanthanide tag improves integral membrane protein structure prediction.

A single experimental method alone often fails to provide the resolution, accuracy, and coverage needed to model integral membrane proteins (IMPs). Integrating computation with experimental data is a powerful approach to supplement missing structural information with atomic detail. We combine RosettaNMR with experimentally-derived paramagnetic NMR restraints to guide membrane protein structure prediction.

Targeting biased signaling by PAR1: function and molecular mechanism of parmodulins.

The G protein-coupled receptor (GPCR) protease-activated receptor 1 (PAR1) is a therapeutic target that was originally pursued with the aim of restricting platelet activation and the burden of cardiovascular diseases. In clinical studies, the use of orthosteric PAR1 inhibitors was associated with an increased risk of hemorrhage, including intracranial hemorrhage. Because (1) PAR1 is expressed by various cell types, including endothelial cells, (2) conveys in mice a physiological indispensable function for vascular development during embryogenesis, and (3) is subject to biased signaling dependent on the activating proteases, orthosteric PAR1 inhibition may be associated with unwanted side effects.

Structural basis of the activation of PPARγ by the plasticizer metabolites MEHP and MINCH.

Di-2-ethylhexyl phthalate (DEHP) and its substitute 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) are widely used as plasticizers but may have adverse health effects. Via hydrolysis of one of the two ester bonds in the human body, DEHP and DINCH form the monoesters MEHP and MINCH, respectively. Previous studies demonstrated binding of these metabolites to PPARγ and the induction of adipogenesis via this pathway.

A transaminase with β-activity from for the production of enantiopure β-amino acids.

Enantioselective transamination of amino acids is a great challenge in biotechnology as suitable enzymes with wide substrate spectrum are rare. Here, we present a new transaminase from (VboTA, ω-transaminase) which is specific for β-amino acids. The amino acid sequence of VboTA is similar to an ω-transaminase from , for which a crystal-structure is available.

Cutinase ACut2 from for the Selective Desymmetrization of the Symmetric Diester Diethyl Adipate to the Monoester Monoethyl Adipate.

Monoethyl adipate (MEA) is a highly valuable monoester for activating resistance mechanisms and improving protective effects in pathogen-attacked plants. The cutinase ACut2 from the non-conventional yeast was used for its synthesis by the desymmetrization of dicarboxylic acid diester diethyl adipate (DEA). Up to 78% MEA with 19% diacid adipic acid (AA) as by-product could be synthesized by the unpurified ACut2 culture supernatant from the overexpression strain.

Molecular Simulations Reveal Distinct Energetic and Kinetic Binding Properties of [F]PI-2620 on Tau Filaments from 3R/4R and 4R Tauopathies.

Tauopathies are a class of neurodegenerative disorders characterized by the accumulation of tau protein filaments in the brain. On the basis of isoforms with three or four microtubule-binding repeats (3R or 4R) that constitute tau filaments, tauopathies can be divided into 3R, 4R, and 3R/4R tauopathies. [F]PI-2620 is a tau-positron emission tomography (PET) tracer that detects tau filaments in the 3R/4R tauopathy Alzheimer's disease (AD) and the 4R tauopathies corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) with differential binding characteristics.

Characterization of Catechol-1,2-Dioxygenase (Acdo1p) From and Investigation of Its Role in the Catabolism of Aromatic Compounds.

Gallic acid, protocatechuic acid, catechol, and pyrogallol are only a few examples of industrially relevant aromatics. Today much attention is paid to the development of new microbial factories for the environmentally friendly biosynthesis of industrially relevant chemicals with renewable resources or organic pollutants as the starting material. The non-conventional yeast, , possesses attractive properties for industrial bio-production processes such as thermo- and osmotolerance.

A new lipase (Alip2) with high potential for enzymatic hydrolysis of the diester diethyladipate to the monoester monoethyladipate.

Several putative lipase genes from the genome of the yeast Blastobotrys (Arxula) raffinosifermentans (adeninivorans) LS3 were overexpressed in the yeast itself and screened for the desymmetrization of the dicarboxylic acid diester diethyl adipate (DEA) into the monoester monoethyl adipate (MEA). MEA can serve as a monomeric spacer group for functional polymers used in medical chemistry and dental applications. The selected lipase Alip2-c6hp was intracellularly located.

The Jacalin-Related Lectin HvHorcH Is Involved in the Physiological Response of Barley Roots to Salt Stress.

Salt stress tolerance of crop plants is a trait with increasing value for future food production. In an attempt to identify proteins that participate in the salt stress response of barley, we have used a cDNA library from salt-stressed seedling roots of the relatively salt-stress-tolerant cv. Morex for the transfection of a salt-stress-sensitive yeast strain ( YSH818 mutant).

Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR.

Labeling of biomolecules with a paramagnetic probe for nuclear magnetic resonance (NMR) spectroscopy enables determining long-range distance restraints, which are otherwise not accessible by classically used dipolar coupling-based NMR approaches. Distance restraints derived from paramagnetic relaxation enhancements (PREs) can facilitate the structure determination of large proteins and protein complexes. We herein present the site-directed labeling of the large oligomeric bacterial DnaB helicase from Helicobacter pylori with cysteine-reactive maleimide tags carrying either a nitroxide radical or a lanthanide ion.