Journal: Biomolecules

Article Title: A Simplified Strategy for Nanobody Production and Use Based on Functional GST-Nanobody Fusion Proteins

doi: 10.3390/biom16020306

Figure Lengend Snippet: ITC analysis of GST-nanobody binding to fluorescent proteins. ( A , B ) Representative ITC thermograms ( top ) and integrated binding isotherms ( bottom ) for GST-nb-GFP titrated into GFP ( A ), and GST-nb-mCherry titrated into mCherry ( B ). Stoichiometry (N) and dissociation constants ( K d ) are reported as mean ± SD ( n = 3).

Article Snippet: The buffer of purified recombinant GFP, GST-nb-GFP, mCherry, and GST-nb-mCherry was exchanged into ITC buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, pH 7.4) using 7K MWCO Zeba TM spin desalting columns (ThermoFisher Scientific).

Techniques: Binding 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