Journal: bioRxiv

Article Title: Exploring Meiosis in Brown Algae: Meiotic Axis Proteins in the model brown alga Ectocarpus

doi: 10.1101/2024.12.18.629156

Figure Lengend Snippet: (A) Upper panel - coomassie stained SDS-PAGE gel of a final size exclusion purification of ecHOP1-HORMA. Lower panel - anti-Strep-II western blot of the same fractions. The corresponding chromatogram can be found in SEC-MALS of ecHOP1-HORMA run on a Superdex 200 5/150 column. Blue trace shows the absorbance at 280 nm in arbitrary units (AU). The gray trace shows the molecular mass measurement. The main peak corresponds to a measured MW of 34.09 kDa. The second peak has a mass of 57.5 kDa. (C-E) Predicted alignment error (PAE) plots of AlphaFold2 predictions of ecHOP1-HORMA with CM1-1, CM2-1 and CM1-2. F) Aligned ecHOP1 closure motifs with the conserved arginine residues highlighted with yellow triangles. G) Pull-Down assays with the WT and mutant (R/A) CMs of ecHOP1. 2xStrepII-ecHOP1-HORMA was used as bait for the prey MBP-tagged CMs. In addition MBP-CMs were used alone as a control for background binding to the Streptactin beads. H-J) Isothermal titration calorimetry (ITC). 350 µM of indicated MBP-tagged closure motif was titrated against 25 µM of ecHOP1-HORMA in the cell. Buffer-buffer controls were run and subtracted from the experimental data to yield the heats shown. Binding curves were fitted in the software and the determined K D is shown. K) AlphaFold2 model of a complex of ecHOP1-isoform1 (with the N-terminal HORMA domain removed), and two copies of ecHOP1-isoform2. The model is coloured as elsewhere, but the isoform2 HORMA domains are coloured in teal. In the PAE plot the CMs are highlighted with the colours as in the cartoon.

Article Snippet: All protein samples were extensively dialyzed in ITC buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA) in Slide-A-Lyzer™ Dialysis Devices, 3.5K MWCO (ThermoFisher) at 4 °C O/N.

Techniques: Staining, SDS Page, Purification, Western Blot, Mass Measurement, Mutagenesis, Control, Binding Assay, Isothermal Titration Calorimetry, Software

Journal: PLoS ONE

Article Title: Structural and Functional Characterization of Cargo-Binding Sites on the μ4-Subunit of Adaptor Protein Complex 4

doi: 10.1371/journal.pone.0088147

Figure Lengend Snippet: Isothermal titration calorimetry of the APP ENPTYKFFEQ peptide with recombinant C-terminal domain of wild-type μ4 ( A ), μ4-D190A ( B ), or μ4-R283D ( C ). The stoichiometry (N) and K d for the interaction of the ENPTYKFFEQ peptide with either μ4-WT or μ4-D190A are expressed as the mean ± SEM (n = 3). Because the interaction of the ENPTYKFFEQ peptide with μ4-R283D is undetectable the stoichiometry and K d were not determined (N/D).

Article Snippet: Recombinant μ4 C-terminal variants were dialyzed overnight at 4°C against excess ITC buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl), and an APP peptide (ENPTYKFFEQ), a CD63 peptide (SGYEVM), or a TGN38 peptide (SDYQRL; New England Peptide, Gardner, MA) were also prepared in ITC buffer.

Techniques: Isothermal Titration Calorimetry, Recombinant

Journal: BMC Complementary Medicine and Therapies

Article Title: Evaluation of antimicrobial activity of the hydrolate of Coridothymus capitatus (L.) Reichenb. fil. (Lamiaceae) alone and in combination with antimicrobial agents

doi: 10.1186/s12906-020-2877-x

Figure Lengend Snippet: Fractional inhibitory concentration (FIC) and FIC indices (FICI) of ITC-hydrolate of C. capitatus pairs against Candida strains

Article Snippet: TC stock solution was dissolved in phosphate buffered solution, pH 7 (PBS; Sigma-Aldrich), whereas ITC stock solutions in dimethylsulfoxide (100%; DMSO; Sigma-Aldrich), and then stored at − 20 °C.

Techniques: Concentration Assay

Journal: Biomolecules

Article Title: Distinct Calcium Binding and Structural Properties of Two Centrin Isoforms from Toxoplasma gondii

doi: 10.3390/biom10081142

Figure Lengend Snippet: Ca 2+ binding to TgCEN1-C and TgCEN2-C. ( A , B ) Representative Ca 2+ titration of apo-TgCEN1-C ( A ) and apo-TgCEN2-C ( B ) as monitored by ITC. Representative raw heat-power changes (upper panels) and integrated binding isotherms (bottom panels). A first injection of 0.5 μL was made and then the first data point was removed from data fitting. Curve fitting was performed by considering the two binding sites model for TgCEN1-C and the one site model for TgCEN2-C. The protein concentration was 70 μM and 100 μM, for apo-TgCEN1-C and apo-TgCEN2-C, respectively. ( C ) The downfield region of the 1 H- 15 N HSQC NMR spectra of TgCEN1-C recorded as a function of increasing Ca 2+ concentration. The molar ratio of Ca 2+ :protein in each case was 0.9, 1.5, 4, and 10 (from left to right).

Article Snippet: Decalcified ITC buffer (50 mM Tris-HCl, 150 mM KCl pH 7.5) was prepared by processing with Chelex 100 resin (BioRad, Hercules, CA, USA) [ , ].

Techniques: Binding Assay, Titration, Injection, Protein Concentration, Concentration Assay

Journal: bioRxiv

Article Title: Development of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic strategy for Alzheimer’s disease

doi: 10.1101/2023.05.22.541727

Figure Lengend Snippet: ( A ) Sequences of phage display peptides. Underlined region corresponds to the variable region within the phage-derived sequence. ( B ) Crystal structure of FERM domain of MSN bound to the C3P-pd peptide. FERM subdomains are displayed as in 1B . C3P-pd peptide (magenta) binds in a pocket between the F1 and F3 subdomains. ( C ) Close-up view of C3P-pd peptide bound to MSN. Peptide intramolecular H-bonds are shown (purple dashed lines). Sidechains of MSN residues interacting with the peptide are displayed, together with their bonding to the peptide (gold dashed lines). ( D ) Superimposed F3 subdomains of MSN from apo (cyan) and C3P-pd bound (light yellow) structures. C3P-pd peptide binding causes a 2.3 Å movement of the beta sheet away from the alpha helix in the MSN F3 lobe, measured from H288 and R246 carbonyl carbon atoms. ( E ) Crystal structure of FERM domain of MSN bound to both C3P-pd and C3S1-pd peptides. ( F ) Close-up of C3S1-pd binding to F3 lobe of MSN. H-bond contacts are shown (green dashed lines). ( G ) Space-filling model of MSN FERM domain, showing C3P-pd (magenta) and CD44 (cyan) binding relative to proposed PIP 2 binding pocket (PIP 2 -BP; blue positively-charged surface). ( H, I, J ) MSN TR-FRET inhibition assays, with acceptor fluorophore conjugated to either ( H ) CD44, ( I ) C3P-pd, or ( J ) C3S1-pd peptides. Unlabelled peptides were used as competitors (C3S1-pd, blue circle; C3P-pd, red square; CD44, green triangle). ( K ) IC 50 values derived from H, I , and J . Asterisk denotes stimulatory rather than inhibitory values ( L ) Binding properties MSN association to C3P-pd and C3S1-pd peptides, measured by ITC. See Figure S3 for thermograms and corresponding fitted curves. K D =dissociation constant, N=stoichiometry, ΔH =enthalpy.

Article Snippet: MSN was buffer exchanged (NAP-5 column; GE Healthcare Life Sciences) and diluted to 20 µM in ITC buffer (50 mM HEPES, pH 7.4, 200 mM NaCl, and 0.5 mM TCEP).

Techniques: Derivative Assay, Sequencing, Binding Assay, Inhibition