Measuring Bacterial Flagellar Motor Dynamics via a Bead Assay.

The bacterial flagellar motor (BFM) is a rotary molecular machine that drives critical bacterial processes including motility, chemotaxis, biofilm formation, and infection. For over two decades, the bead assay, which measures the rotation of a microparticle attached to the flagellum of a surface-attached bacterium, has been instrumental in deciphering the motor's biophysical mechanisms. This technique has not only quantified the rotational speed and frequency of directional switching as a function of the viscous load on the flagellum but has also revealed the BFM's capacity for mechanosensitive speed modulation, adapting to environmental conditions.

Determining the Relative Affinity of ORPs for Lipid Ligands Using Fluorescence and Thermal Shift Assays.

Lipid transfer proteins (LTPs) are specialized proteins that convey specific lipids across the cytosol to regulate the lipid composition of organelles and the plasma membrane. Quantifying to which extent these LTPs recognize and transfer various lipid species and subspecies is of prime interest to define their cellular role(s). Here, we describe how to measure in vitro the relative affinity of Osh6p, a yeast phosphatidylserine (PS)/phosphatidylinositol 4-phosphate (PI(4)P) exchanger belonging to the oxysterol-binding protein(OSBP)-related protein (ORP) family, for PS and phosphoinositide subspecies.

Structure of the aminoterminal domain of the birnaviral multifunctional VP3 protein and its unexplored critical role.

To overcome their limited genetic capacity, numerous viruses encode multifunctional proteins. The birnavirus VP3 protein plays key roles during infection, including scaffolding of the viral capsid during morphogenesis, recruitment, and regulation of the viral RNA polymerase, shielding of the double-stranded RNA genome and targeting of host endosomes for genome replication, and immune evasion. The dimeric form of VP3 is critical for these functions.

Site-Specific Incorporation of Fluorinated Prolines into Proteins and Their Impact on Neighbouring Residues.

The incorporation of fluorinated amino acids into proteins provides new opportunities to study biomolecular structure-function relationships in an elegant manner. The available strategies to incorporate the majority of fluorinated amino acids are not site-specific or imply important structural modifications. Here, we present a chemical biology approach for the site-specific incorporation of three commercially available C-modified fluoroprolines that has been validated using a non-pathogenic version of huntingtin exon-1 (HttExon-1).

Towards the design of ligands of the internal pocket TEADs C-terminal domain.

The Hippo pathway controls in organ size and tissue homeostasis through regulating cell growth, proliferation and apoptosis. Phosphorylation of the transcription co-activator YAP (Yes associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif) regulates their nuclear import and therefore their interaction with TEAD (Transcriptional Enhanced Associated Domain). YAP, TAZ and TEADs are dysregulated in several solid cancers making YAP/TAZ-TEAD interaction a new anti-cancer target.

Microbial biosensors for diagnostics, surveillance and epidemiology: Today's achievements and tomorrow's prospects.

Microbial biosensors hold great promise for engineering high-performance, field-deployable and affordable detection devices for medical and environmental applications. This review explores recent advances in the field, highlighting new sensing strategies and modalities for whole-cell biosensors as well as the remarkable expansion of microbial cell-free systems. We also discuss improvements in robustness that have enhanced the ability of biosensors to withstand the challenging conditions found in biological samples.

Structural Insights Into Thyroid Hormone Receptors.

Thyroid hormone receptors (TRs) are essential components of the endocrine system, mediating the cellular effects of thyroid hormones. The 2 TR genes, THRA and THRB, encode 4 isoforms, with TRα1 and TRβ1 being the most prevalent. TRs are ligand-dependent transcription factors and members of the nuclear receptor superfamily, indispensable for human growth, development, and metabolism.

Biased activation of the vasopressin V2 receptor probed by molecular dynamics simulations, NMR and pharmacological studies.

G protein-coupled receptors (GPCRs) control critical cell signaling. Their response to extracellular stimuli involves conformational changes to convey signals to intracellular effectors, among which the most important are G proteins and β-arrestins (βArrs). Biased activation of one pathway is a field of intense research in GPCR pharmacology.

Weighted families of contact maps to characterize conformational ensembles of (highly-)flexible proteins.

Motivation: Characterizing the structure of flexible proteins, particularly within the realm of intrinsic disorder, presents a formidable challenge due to their high conformational variability. Currently, their structural representation relies on (possibly large) conformational ensembles derived from a combination of experimental and computational methods. The detailed structural analysis of these ensembles is a difficult task, for which existing tools have limited effectiveness.

Molecular model of a bacterial flagellar motor reveals a "parts-list" of protein adaptations to increase torque.

One hurdle to understanding how molecular machines work, and how they evolve, is our inability to see their structures . Here we describe a minicell system that enables cryogenic electron microscopy imaging and single particle analysis to investigate the structure of an iconic molecular machine, the bacterial flagellar motor, which spins a helical propeller for propulsion. We determine the structure of the high-torque motor including the subnanometre-resolution structure of the periplasmic scaffold, an adaptation essential to high torque.

Split Reporters Facilitate Monitoring of Gene Expression and Peptide Production in Linear Cell-Free Transcription-Translation Systems.

Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable the rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present reporter systems based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in TXTL.

A PRE loop at the dac locus acts as a topological chromatin structure that restricts and specifies enhancer-promoter communication.

Three-dimensional (3D) genome folding has a fundamental role in the regulation of developmental genes by facilitating or constraining chromatin interactions between cis-regulatory elements (CREs). Polycomb response elements (PREs) are a specific kind of CRE involved in the memory of transcriptional states in Drosophila melanogaster. PREs act as nucleation sites for Polycomb group (PcG) proteins, which deposit the repressive histone mark H3K27me3, leading to the formation of a class of topologically associating domain (TAD) called a Polycomb domain.

What Structural Biology Tells Us About the Mode of Action and Detection of Toxicants.

The study of the adverse effects of chemical substances on living organisms is an old and intense field of research. However, toxicological and environmental health sciences have long been dominated by descriptive approaches that enable associations or correlations but relatively few robust causal links and molecular mechanisms. Recent achievements have shown that structural biology approaches can bring this added value to the field.

Detection and Analysis of Short Linear Motif-Based Protein-Protein Interactions with SLiMAn2 Web Server.

Interactomics is bringing a deluge of data regarding protein-protein interactions (PPIs) which are involved in various molecular processes in all types of cells. However, this information does not easily translate into direct and precise molecular interfaces. This limits our understanding of each interaction network and prevents their efficient modulation.

The TOPOVIBL meiotic DSB formation protein: new insights from its biochemical and structural characterization.

The TOPOVIL complex catalyzes the formation of DNA double strand breaks (DSB) that initiate meiotic homologous recombination, an essential step for chromosome segregation and genetic diversity during gamete production. TOPOVIL is composed of two subunits (SPO11 and TOPOVIBL) and is evolutionarily related to the archaeal TopoVI topoisomerase complex. SPO11 is the TopoVIA subunit orthologue and carries the DSB formation catalytic activity.

fate of Chikungunya virus replication organelles.

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation.

Mechanical characterization of regenerating Hydra tissue spheres.

Hydra vulgaris, long known for its remarkable regenerative capabilities, is also a long-standing source of inspiration for models of spontaneous patterning. Recently it became clear that early patterning during Hydra regeneration is an integrated mechanochemical process whereby morphogen dynamics is influenced by tissue mechanics. One roadblock to understanding Hydra self-organization is our lack of knowledge about the mechanical properties of these organisms.

SLiMAn 2.0: meaningful navigation through peptide-protein interaction networks.

Among the myriad of protein-protein interactions occurring in living organisms, a substantial amount involves small linear motifs (SLiMs) recognized by structured domains. However, predictions of SLiM-based networks are tedious, due to the abundance of such motifs and a high portion of false positive hits. For this reason, a webserver SLiMAn (Short Linear Motif Analysis) was developed to focus the search on the most relevant SLiMs.

Genome-wide association analysis uncovers rice blast resistance alleles of Ptr and Pia.

A critical step to maximize the usefulness of genome-wide association studies (GWAS) in plant breeding is the identification and validation of candidate genes underlying genetic associations. This is of particular importance in disease resistance breeding where allelic variants of resistance genes often confer resistance to distinct populations, or races, of a pathogen. Here, we perform a genome-wide association analysis of rice blast resistance in 500 genetically diverse rice accessions.

TDP-43 proteinopathy in ALS is triggered by loss of ASRGL1 and associated with HML-2 expression.

TAR DNA-binding protein 43 (TDP-43) proteinopathy in brain cells is the hallmark of amyotrophic lateral sclerosis (ALS) but its cause remains elusive. Asparaginase-like-1 protein (ASRGL1) cleaves isoaspartates, which alter protein folding and susceptibility to proteolysis. ASRGL1 gene harbors a copy of the human endogenous retrovirus HML-2, whose overexpression contributes to ALS pathogenesis.

How total mRNA influences cell growth.

Experimental observations tracing back to the 1960s imply that ribosome quantities play a prominent role in determining a cell's growth. Nevertheless, in biologically relevant scenarios, growth can also be influenced by the levels of mRNA and RNA polymerase. Here, we construct a quantitative model of biosynthesis providing testable scenarios for these situations.

The structural landscape and diversity of Pyricularia oryzae MAX effectors revisited.

Magnaporthe AVRs and ToxB-like (MAX) effectors constitute a family of secreted virulence proteins in the fungus Pyricularia oryzae (syn. Magnaporthe oryzae), which causes blast disease on numerous cereals and grasses. In spite of high sequence divergence, MAX effectors share a common fold characterized by a ß-sandwich core stabilized by a conserved disulfide bond.

Free-Standing DNA Origami Superlattice to Facilitate Cryo-EM Visualization of Membrane Vesicles.

Technological breakthroughs in cryo-electron microscopy (cryo-EM) methods open new perspectives for highly detailed structural characterizations of extracellular vesicles (EVs) and synthetic liposome-protein assemblies. Structural characterizations of these vesicles in solution under a nearly native hydrated state are of great importance to decipher cell-to-cell communication and to improve EVs' application as markers in diagnosis and as drug carriers in disease therapy. However, difficulties in preparing holey carbon cryo-EM grids with low vesicle heterogeneities, at low concentration and with kinetic control of the chemical reactions or assembly processes, have limited cryo-EM use in the EV study.