atto647n labeled gamma atp  (Jena Bioscience)

Journal: bioRxiv

Article Title: Active regulation of the epidermal growth factor receptor by the membrane bilayer

doi: 10.1101/2025.08.14.670284

Figure Lengend Snippet: (a) Full-length EGFR in nanodiscs with atto647N γ ATP (red sphere) and snap surface 488 or snap surface 594 (green sphere). (b) Extent of ATP binding in different anionic environments quantified using the intensity of atto 647N (top) band normalized by the amount of EGFR produced as extracted from the intensity of ss488 band (center) as a function of negatively charged POPS lipids (bottom) in the absence (purple) and presence (orange) of EGF. Error bars from three independent biological replicates. (c) EGFR intracellular domain indicating ATP binding site-C-terminus distance and ATP-lipid contacts measured from molecular dynamics simulations. (d) Accessibility of ATP binding site quantified through the contact number between the ATP binding site and lipids in the absence (purple) and presence (orange) of EGF. smFRET donor lifetime distributions with atto647N γ ATP as acceptor and snap surface 594 as donor in (e) neutral (0% POPS) and (f) 30% anionic lipids (30% POPS) without EGF (top); with 1 µ M EGF (bottom). Probability distributions of the distance between residue 721, the closest residue to the ATP binding site, and EGFR C-terminus for (g) neutral (0% POPS) and (h) 30% anionic lipids (30% POPS). Dotted lines indicate the medians on all histograms with corresponding distances on upper x-axis.

Article Snippet: 1 μ M of snap surface 488 (New England Biolabs) and atto647N labeled gamma ATP (Jena Bioscience) was added after cell-free expression and incubated at 30 o , 300 rpm for 60 minutes.

Techniques: Binding Assay, Produced, Residue

Journal: BMC Plant Biology

Article Title: Transcript profiling of plastid ferrochelatase two mutants reveals that chloroplast singlet oxygen signals lead to global changes in RNA profiles and are mediated by Plant U-Box 4

doi: 10.1186/s12870-025-06703-7

Figure Lengend Snippet: Jasmonic acid biosynthesis and signaling are induced in fc2 under cycling light conditions. The impact of singlet oxygen ( 1 O 2 ) on jasmonic acid (JA) biosynthesis and signaling was assessed in constant light (24 h) and 16 h light/8 h dark diurnal (cycling) light conditions. ( a ) Schematic illustration of the JA biosynthesis pathway, with relative expression (compared to wt in the same light condition) of 11 key JA biosynthesis and signaling genes. Expression data is from the DESeq2 analysis of the included RNA-seq data set. Error bars in the graph represent standard error (ifcSE) estimated by DESeq2 (**, padj < 0.01; ***, padj < 0.001). In chloroplasts, Defective In Anther Dehiscence 1 (DAD1) catalyzes the conversion of galactolipids into α-linolenic acid (α-LeA), which is converted into 12-oxo-phytodienoic acid (OPDA) by a series of enzymes; 13-Lipoxygenase (LOX), Allene Oxide Synthase (AOS), and Allene Oxide Cyclase (AOC). Subsequently, OPDA is exported out of the chloroplast via the channel protein JASSY. Inside the peroxisome, OPDA is reduced by the OPDA reductase 3 (OPR3) and shortened in the carboxylic acid side chain by β-oxidation enzymes (ACX1) into JA. In the cytosol, Jasmonate Resistant 1 (JAR1) conjugates isoleucine to JA converting it into JA-Ile. Jasmonate Transporter 1 (JAT1) transports JA-Ile into the nucleus. In response to stressed conditions, JA-Ile attach with the COI1-SCF (coronatine insensitive1- Skp1-Cul1-F-box), to promote ubiquitination and degradation of jasmonic acid repressors JAZ and JAV1, thereby activating JA response genes. ( b ) Graph showing JA content (ng/g fresh weight (FW)) measured in whole rosettes from plants grown for 19 days constant (24 h) light conditions or 17 days in 24 h light conditions and two days of 16 h light/8 h dark diurnal (cycling) light conditions. Values are means ± SEM ( n = 3 biological replicates). Statistical analyses in b were performed using one-way ANOVA tests, and the different letters above the bars indicate significant differences within data sets determined by Tukey–Kramer post-tests ( P ≤ 0.05). Separate analyses were performed for the different light treatments, and the significance of cycling light treatment is denoted by letters with a prime symbol (ʹ). ( c ) Heatmap showing relative expression of 58 genes from gene ontology term “JA mediated signaling pathway” (GO:0009867). The blue and red colors correspond to low and high gene expression, respectively

Article Snippet: Polyclonal antibodies for NAD(P)H dehydrogenase subunit 45 (NDH45), Thylakoid Membrane Cytochrome B6 Protein (PetB), ATP Synthase Gamma Chain (AtpC), and Rubisco large subunit (RbcL) were purchased from PhytoAB (California, USA).

Techniques: Expressing, RNA Sequencing, Ubiquitin Proteomics, Gene Expression

Journal: Nature Communications

Article Title: Deciphering the allosteric regulation of mycobacterial inosine-5′-monophosphate dehydrogenase

doi: 10.1038/s41467-024-50933-6

Figure Lengend Snippet: a Side and top views of an Msm IMPDH octamer: the first tetramer is depicted in blue and gold, with its opposite tetramer in green and grey; the Msm IMPDH monomer is highlighted in saturated colours. b Structural details of the highlighted Msm IMPDH monomer. The catalytic domain is depicted in blue and the CBS domain in gold. The flexible loops of the Cys-loop (residues 320–326) are shown in orange, the finger (residues 391–404) in red, the flap (residues 405–450) in purple, and the IMP molecule in green. The two fingers and distal parts of the flaps form a binding interface between the two opposite protomers. c Four ATP molecules (in red) bound within Site 1 and 2 in a complex with four Mg 2+ ions (in green) form an interface between two neighbouring CBS domains.

Article Snippet: Our procedure involved the preparation of a 10-ml mixture containing 50 μM Msm IMPDH, 6 nM radiolabelled ATP [γ-33P] (0.4 mCi; Hartmann Analytic) in 50 mM HEPES (pH 7.5), 200 mM KCl, 1 mM MgCl 2 , and 0.5 mM TCEP.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Deciphering the allosteric regulation of mycobacterial inosine-5′-monophosphate dehydrogenase

doi: 10.1038/s41467-024-50933-6

Figure Lengend Snippet: a Heat map of apo Msm IMPDH (constructed from 221 generated peptides) shows the deuteration levels of each amino acid residue in Msm IMPDH obtained at 4 °C over a time course of 2–120 s. Each line represents one time point (2 s, 5 s, 10 s, 20 s, and 2 min). The degree of deuteration is represented by the relative fractional uptake, scaled in rainbow colours from the minimum to the maximum observed uptake. The residues with no sequence coverage are shown as white gaps in the heat map. The sequence coverage is depicted by purple lines above the sequence. Differential heat maps of Msm IMPDH under conditions of ( b ) apo vs ATP, and ( c ) ATP–IMP vs ATP–GTP–IMP. Differences in the relative fractional uptake are displayed in a blue–white–red colour scale: residues with decreased accessibility are in blue, and residues with increased HDX are in red. d The ΔHDX changes of ATP–IMP vs ATP–GTP–IMP at 10 s are mapped on the Msm IMPDH monomer structure. Red indicates elevated deuteration, and blue less extensive deuteration of the given region. Hinge regions with residues involved in the binding of GTP (Arg108 and Lys222) are outlined by separate black frames. Deuterium uptake plots show the evolution in the deuteration of four representative peptides in the hinge region. In the graphs, each line represents one protein state, and each point represents one time point over a time course of 2–120 s. For comparison, the protein states are displayed by the same symbols. The drops observed in the HDX rate are displayed in light- and dark-blue colours.

Article Snippet: Our procedure involved the preparation of a 10-ml mixture containing 50 μM Msm IMPDH, 6 nM radiolabelled ATP [γ-33P] (0.4 mCi; Hartmann Analytic) in 50 mM HEPES (pH 7.5), 200 mM KCl, 1 mM MgCl 2 , and 0.5 mM TCEP.

Techniques: Construct, Generated, Residue, Sequencing, Binding Assay, Comparison

Journal: Nature Communications

Article Title: Deciphering the allosteric regulation of mycobacterial inosine-5′-monophosphate dehydrogenase

doi: 10.1038/s41467-024-50933-6

Figure Lengend Snippet: a The binding of IMP (in green) induces movement of the Cys-loop (residues 320–326, in orange) from an open conformation to a closed conformation. b The initial part of the flap loop (residues 405–415, in purple) interacts with IMP through residues Tyr405, Met408, and Gly409. The contacts are depicted by the grey dotted lines; each number indicates the distance in Å. c IMP binding induces a movement of about 3.5 Å of the entire finger loop (residues 391–404 in red), as indicated by the red arrows. d The two-dimensional representation shows the contacts between IMP and residues forming the Msm IMPDH active site. e Changes to the finger (in red), flap (in purple), and Cys-loop (in orange) after IMP binding (green spheres) lead to extensive rearrangement of the tetramer–tetramer interface, enabling octamer expansion. For consistency, the ATP-bound Msm IMPDH structure was chosen as the model for the compressed conformation across all panels (grey cartoon in a – c ). f SAXS profiles of 56 µM Msm IMPDH show the effect of nucleotides on the quaternary structure. For ease of visualization, plots are conveniently displaced along the y axis to represent the three obtained conformations: (green) tetramers induced by 10 mM IMP; (orange) compressed octamers in the apo state, (red) with 10 mM GTP, and (blue) with 10 mM ATP; and (purple) extended octamers induced by 10 mM ATP and IMP. Dashed lines show the theoretical SAXS profiles calculated from the respective cryo-EM structures fitted to the experimental scattering curves. The curve fitted to the IMP dataset (marked by *) represents a 75:25% mixture of tetramers and octamers, while the fit for the ATP + IMP dataset (marked by **) indicates a 38:62% mixture of compressed and extended conformations, as calculated using the OLIGOMER program . g Mass photometry profiles of 20 nM Msm IMPDH in its apo state and in the presence of 5 mM ATP and/or IMP reveal two distinct peaks. The observed particle masses of the first and second peaks correspond with the Msm IMPDH tetramer (213.2 kDa) and octamer (426.4 kDa).

Article Snippet: Our procedure involved the preparation of a 10-ml mixture containing 50 μM Msm IMPDH, 6 nM radiolabelled ATP [γ-33P] (0.4 mCi; Hartmann Analytic) in 50 mM HEPES (pH 7.5), 200 mM KCl, 1 mM MgCl 2 , and 0.5 mM TCEP.

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Deciphering the allosteric regulation of mycobacterial inosine-5′-monophosphate dehydrogenase

doi: 10.1038/s41467-024-50933-6

Figure Lengend Snippet: a ATP binds at Site 2 with the base in anti conformation relative to the ribose. b The GTP molecule binds with the base in syn conformation. The other nucleotide is shown as thin lines in the background for comparison of a and b. The contacts with Arg108 and Lys222 (in gold) are depicted by grey dotted lines; each number indicates the distance in Å. c The ppGpp molecule binds at a separate binding site in the CBS domain. d The hinge regions are flexible in the case of ATP binding (in gold), while the GTP locks the hinges in a fixed position (in grey). The side chains of Arg108 and Lys222 are depicted as sticks. e The mutation of Arg108A and Lys222A strongly reduces the inhibition of Msm IMPDH by ppGpp and GTP. NAD + and IMP substrates were fixed at concentrations of 2 mM and 100 µM, respectively; ATP was fixed at 500 µM. The relative velocity value was calculated as the ratio of the initial velocity of the reaction to the control reaction of the corresponding enzyme ( n = 3). Data are presented as mean values with error bars representing the SD.

Article Snippet: Our procedure involved the preparation of a 10-ml mixture containing 50 μM Msm IMPDH, 6 nM radiolabelled ATP [γ-33P] (0.4 mCi; Hartmann Analytic) in 50 mM HEPES (pH 7.5), 200 mM KCl, 1 mM MgCl 2 , and 0.5 mM TCEP.

Techniques: Comparison, Binding Assay, Mutagenesis, Inhibition, Control

Journal: bioRxiv

Article Title: PLK-1 regulates MEX-1 polarization in the C. elegans zygote

doi: 10.1101/2024.07.26.605193

Figure Lengend Snippet: MEX-1 is a PLK1 substrate. A. Schematic of MEX-1 including the position of the RNA-binding zinc finger domains ZF1 and ZF2. Black circles in the panel below shows the position of predicted PLK1 phos-phorylation sites and phosphorylated residues detected by phospho-mass spectrometry of embryo lysates ( in vivo ; Offenburger 2017) or following in vitro kinase assays (as in panel B). The position of alanine substitutions in MEX-1 alleles used in this study are shown. Note that alleles in the N2, MEX-1::OLLAS and MEX-1::GFP backgrounds were made independently. B. In vitro kinase assay with hPLK1 and the indicated substrates. Top panel: Phosphorylation was detected by western blot using an anti-thiophosphate ester antibody, which recognizes alkylated ATP-γS. Bottom panel: total protein was detected by Coommassie Brilliant Blue (CBB) staining. The position of human hPLK1 is indicated to the right and the position of molecular weight markers (not shown) is indicated to the left. Schematic of the recombinant MEX-1 constructs used are shown above. C. Quantification hPLK1 phosphorylation of over time. Three replicates were normalized to the background (equals 0) the final MBP:MEX-1(aa1-299):6XHis value (equals 1) within each experiment and averaged. D. Top: Fluorescence micrographs of one-cell embryos immunostained using an anti-OLLAS antibody. Bottom: Average fluorescence intensity along the anterior/posterior axis of immunostained embryos. Intensities from the indicated number of embryos were normalized to the anterior end and averaged. E. Ratio of P1/AB fluorescence intensity in 2-cell embryos stained for MEX-1::OLLAS. Each dot indicates an individual embryo. The mean and SEM are shown. F. Total fluorescence intensity of immunostained MEX-1::OLLAS and MEX-1(4A C-term)::OLLAS zygotes. In this and subsequent figures, **** = p < 0.0001; *** = p < 0.001*, ** = p < 0.01, * = p < 0.05., n.s. = not significant.

Article Snippet: Kinase assays were performed at 37°C by diluting 0.2ug of substrate protein into kinase reaction buffer (8 mM MOPS, pH 7.0, 100 μM Zn(OAc) 2 , 10 mM MgCl 2 and protease cocktail (K1010, APExBIO, Houston, TX, USA) containing 2 mM ATP-γS (Abcam) and 375 ng of hPLK1 (EMD Millipore).

Techniques: RNA Binding Assay, Mass Spectrometry, In Vivo, In Vitro, Kinase Assay, Western Blot, Staining, Molecular Weight, Recombinant, Construct, Fluorescence