The BumS sensor phosphatase detects the branched short-chain fatty acids isobutyrate and isovalerate as direct cues for signal transduction.

Two-component signal transduction systems (TCSs) are nearly ubiquitous across bacterial species and enable bacteria to sense and respond to specific cues for environmental adaptation. The BumSR TCS is unusual in that the BumS sensor exclusively functions as a phosphatase rather than a kinase to control phosphorylated levels of its cognate BumR response regulator (P-BumR). We previously found that BumSR directs a response to the short-chain fatty acid butyrate generated by resident microbiota so that identifies ideal lower intestinal niches in avian and human hosts for colonization.

Structures of the multi-domain oxygen sensor DosP: remote control of a c-di-GMP phosphodiesterase by a regulatory PAS domain.

The heme-based direct oxygen sensor DosP degrades c-di-GMP, a second messenger nearly unique to bacteria. In stationary phase Escherichia coli, DosP is the most abundant c-di-GMP phosphodiesterase. Ligation of O to a heme-binding PAS domain (hPAS) of the protein enhances the phosphodiesterase through an allosteric mechanism that has remained elusive.

Structures of the multi-domain oxygen sensor DosP: remote control of a c-di-GMP phosphodiesterase by a regulatory PAS domain.

The heme-based direct oxygen sensor DosP degrades c-di-GMP, a second messenger nearly unique to bacteria. In stationary phase , DosP is the most abundant c-di-GMP phosphodiesterase. Ligation of O to a heme-binding PAS domain (hPAS) of the protein enhances the phosphodiesterase through an allosteric mechanism that has remained elusive.

Analysis of Arc/Arg3.1 Oligomerization In Vitro and in Living Cells.

Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation, long-term depression, and synaptic scaling.

Biophysical characterization of the CXC chemokine receptor 2 ligands.

The chemokines of the immune system act as first responders by operating as chemoattractants, directing immune cells to specific locations of inflamed tissues. This promiscuous network is comprised of 50 ligands and 18 receptors where the ligands may interact with the receptors in various oligomeric states i.e.

A new Karyopherin-β2 binding PY-NLS epitope of HNRNPH2 linked to neurodevelopmental disorders.

The HNRNPH2 proline-tyrosine nuclear localization signal (PY-NLS) is mutated in HNRNPH2-related X-linked neurodevelopmental disorder, causing the normally nuclear HNRNPH2 to accumulate in the cytoplasm. We solved the cryoelectron microscopy (cryo-EM) structure of Karyopherin-β2/Transportin-1 bound to the HNRNPH2 PY-NLS to understand importin-NLS recognition and disruption in disease. HNRNPH2 RPGPY is a typical R-X-P-Y motif comprising PY-NLS epitopes 2 and 3, followed by an additional Karyopherin-β2-binding epitope, we term epitope 4, at residues DRP; no density is present for PY-NLS epitope 1.

Biophysical and biochemical studies support TP0094 as a phosphotransacetylase in an acetogenic energy-conservation pathway in Treponema pallidum.

The mechanisms of energy generation and carbon-source utilization in the syphilis spirochete Treponema pallidum have remained enigmatic despite complete genomic sequence information. Whereas the bacterium harbors enzymes for glycolysis, the apparatus for more efficient use of glucose catabolites, namely the citric-acid cycle, is apparently not present. Yet, the organism's energy needs likely exceed the modest output from glycolysis alone.

SViMULATE: a computer program facilitating interactive, multi-mode simulation of analytical ultracentrifugation data.

The ability to simulate sedimentation velocity (SV) analytical ultracentrifugation (AUC) experiments has proved to be a valuable tool for research planning, hypothesis testing, and pedagogy. Several options for SV data simulation exist, but they often lack interactivity and require up-front calculations on the part of the user. This work introduces SViMULATE, a program designed to make AUC experimental simulation quick, straightforward, and interactive.

A new Karyopherin-β2 binding PY-NLS epitope of HNRNPH2 is linked to neurodevelopmental disorders.

Unlabelled: The normally nuclear HNRNPH2 is mutated in -related X-linked neurodevelopmental disorder causing the protein to accumulate in the cytoplasm. Interactions of HNRNPH2 with its importin Karyopherin-β2 (Transportin-1) had not been studied. We present a structure that shows Karyopherin-β2 binding HNRNPH2 residues 204-215, a proline-tyrosine nuclear localization signal or PY-NLS that contains a typical R-X -P-Y motif, RPGPY , followed a new Karyopherin-β2 binding epitope at DRP that make many interactions with Karyopherin-β2 W373.

Oligomer-to-monomer transition underlies the chaperone function of AAGAB in AP1/AP2 assembly.

Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin-binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homodimer.

Self-assembling Peptides with Internal Ionizable Unnatural Amino Acids: A General Approach to pH-responsive Peptide Materials.

Self-assembled peptides are an emerging family of biomaterials that show great promise for a range of biomedical and biotechnological applications. Introducing and tuning the pH-responsiveness of the assembly is highly desirable for improving their biological activities. Inspired by proteins with internal ionizable residues, we report a simple but effective approach to constructing pH-responsive peptide assembly containing unnatural ionic amino acids with an aliphatic tertiary amine side chain.

The feasibility of determining kinetic constants from isothermal titration calorimetry data.

Isothermal titration calorimetry (ITC) has long been established as an excellent means to determine the thermodynamic parameters of biomolecular interactions. More recently, efforts have focused on exploiting the power/time trace (the "thermogram") resulting from ITC experiments to glean kinetic association and dissociation rates for these interactions. The success of such analyses rests on the ability of algorithms to simulate with high accuracy the output of the calorimeter.

A Protein Semisynthesis-Based Strategy to Investigate the Functional Impact of Linker Histone Serine ADP-Ribosylation.

Recently developed chemical and enzyme-based technologies to install serine ADP-ribosylation onto synthetic peptides have enabled new approaches to study poly(ADP-ribose) polymerase (PARP) biology. Here, we establish a generalizable strategy to prepare ADP-ribosylated peptides that are compatible with N-terminal, C-terminal, and sequential protein ligation reactions. Two unique protein-assembly routes are employed to generate full-length linker histone constructs that are homogeneously ADP-ribosylated at known DNA damage-dependent modification sites.

Inhibition of bacterial FMN transferase: A potential avenue for countering antimicrobial resistance.

Antibiotic resistance is a challenge for the control of bacterial infections. In an effort to explore unconventional avenues for antibacterial drug development, we focused on the FMN-transferase activity of the enzyme Ftp from the syphilis spirochete, Treponema pallidum (Ftp_Tp). This enzyme, which is only found in prokaryotes and trypanosomatids, post-translationally modifies proteins in the periplasm, covalently linking FMN (from FAD) to proteins that typically are important for establishing an essential electrochemical gradient across the cytoplasmic membrane.

Modular Design of Supramolecular Ionic Peptides with Cell-Selective Membrane Activity.

The rational design of materials with cell-selective membrane activity is an effective strategy for the development of targeted molecular imaging and therapy. Here we report a new class of cationic multidomain peptides (MDPs) that can undergo enzyme-mediated molecular transformation followed by supramolecular assembly to form nanofibers in which cationic clusters are presented on a rigid β-sheet backbone. This structural transformation, which is induced by cells overexpressing the specific enzymes, led to a shift in the membrane perturbation potential of the MDPs, and consequently enhanced cell uptake and drug delivery efficacy.

Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study.

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.

Measuring the K of Protein-Ligand Interactions Using Microscale Thermophoresis.

Microscale thermophoresis (MST) has become a widely used technique to determine the K or EC of protein-ligand interactions. The method exploits the tendency of macromolecules to migrate along a thermal gradient (i.e.

Osmosensing by WNK Kinases.

With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol (PEG)400 or osmolyte ethylene glycol (EG), and that this activation is opposed by chloride.

Molecular Size Analysis of Recombinant Importin-histone Complexes Using Analytical Ultracentrifugation.

Histones constitute the protein components of nucleosomes. Despite their small sizes, histones do not diffuse through the nuclear pore complex. Instead, they are transported to the nucleus by importins, either alone or in complex with histone chaperones.

Mechanism of karyopherin-β2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X).

Mutations in the RNA-binding protein FUS cause familial amyotropic lateral sclerosis (ALS). Several mutations that affect the proline-tyrosine nuclear localization signal (PY-NLS) of FUS cause severe juvenile ALS. FUS also undergoes liquid-liquid phase separation (LLPS) to accumulate in stress granules when cells are stressed.

Biophysical and Biochemical Characterization of TP0037, a d-Lactate Dehydrogenase, Supports an Acetogenic Energy Conservation Pathway in Treponema pallidum.

A longstanding conundrum in biology concerns how the spirochete generates sufficient energy to fulfill its complex pathogenesis processes during human syphilitic infection. For decades, it has been assumed that the bacterium relies solely on glucose catabolism (via glycolysis) for generation of its ATP. However, the organism's robust motility, believed to be essential for human tissue invasion and dissemination, would require abundant ATP likely not provided by the parsimony of glycolysis.