Microcal itc200 system

A MicroCal iTC200 system (Malvern Panalytical, Almelo, Netherlands) was used to measure enthalpy changes associated with interactions between the YwfG variants and the following yeast cell-wall components: the monosaccharides d-glucose, d-galactose, d-mannose, d-fucose, N-acetyl-d-glucosamine (GlcNAc), and 2-(acetylamino)-2-deoxy-d-galactose (GalNAc) (Sigma-Aldrich); α-1,2-, α-1,3-, α-1,4-, and α-1,6-mannobiose (Dextra Laboratories, Reading, UK); mannan from S. cerevisiae (Sigma-Aldrich); and yeast mannoproteins.
A solution of YwfG variant (100 μM YwfG_28–270, YwfG_28–336, YwfG_28–511, or MubR4 in 20 mM Tris-HCl [pH 7.5]) was placed in a 200-μL calorimeter cell, and test solution (10 mM for monosaccharides, 2 mM for monosaccharides and mannobioses, 10 mg mL⁻¹ for yeast mannan, OD₂₈₀ 1.0 for mannoproteins) was loaded into the injection syringe. The test solution was titrated into the sample cell as a sequence of 20 injections of 2-μL aliquots each. All experiments were performed at 25°C.

Isothermal titration
calorimetry experiments were conducted on a MicroCal iTC200 System
(Malvern Instruments). The proteins were dialyzed against a buffer
containing 500 mM NaCl and 25 mM HEPES (pH 7.4). Protein concentrations
were determined using calculated extinction coefficients at 280 nm
and diluted to the experimental concentration using dialysis buffer.
The APP peptide was placed in the syringe with a 10-fold higher concentration
compared to that of PTB/PARM in the sample cell. During injections,
the sample cell was held at 25 °C. The data were analyzed with
ITC Origin software and fitted to a one-binding site model. The binding
stoichiometry was determined to be between 0.9 and 1.1.

Isothermal titration calorimetry experiments were performed using a Malvern MicroCal iTC200 system at 25°C. Here, the lpWalK homologue smVicK was also used because the lpWalK CA domain was not stable. The smVicK gene fragments encoding the DHp/CA domains (amino acids 196–450) and the CA domain (amino acids 271–450) were cloned in the pET-His vector, and the proteins were expressed and purified as described above. All lpWalK and smVicK samples were prepared in the same buffer consisting of 20 mM Tris–HCl pH 8.0, 5 mM MgCl₂. The sample cell was filled with 50 µM VicK and titrated with the ligands AMPPNP, ADP or AMP at concentrations that were empirically determined to have the best signal-to-noise ratio. The concentrations of AMPPNP and ADP were set to 3 mM and the concentration of AMP was set to 6 mM. The delay time was 60 s for the first titration of 0.4 µl of ligand. The rest of the titrations were completed with 2 µl of ligand per each injection for 20 intervals of 120 s. The stirring speed was 1000 rev min⁻¹. The reference power was 5 µcal s⁻¹. The data were fitted with a fixed 1:1 binding model in the Microcal Analysis Launcher software package. More than 200 iterations were used to reduce the χ² until all values remained constant. The final K_d, ΔH and ΔS values after the fitting are displayed on the plot.

To determine the binding affinities of the discussed compounds for Zn²⁺, isothermal titration calorimetry experiments were conducted on a MicroCal ITC-200 system (Malvern Instruments) at a temperature of 30°C. The compounds, provided as a DMSO stock solution of 10 mM, were diluted in 50 mM HEPES (pH 7.4) and 150 mM NaCl to reach a final nominal concentration of 400 μM (= 4% (v/v) final DMSO concentration). Complete titration of the compounds was typically achieved by injecting 27 x 1.3 μl aliquots of 5 mM ZnCl₂ provided in a matching buffer. The thermodynamic binding parameters were extracted by non-linear regression analysis of the binding isotherms (Microcal Origin version 7.0 software package). A single site binding model was applied, yielding binding enthalpy (ΔH), stoichiometry (n), entropy (ΔS), and association constant (K_a). All experiments have been performed in triplicate to allow for error estimations.

ITC assays were carried out on a MicroCal iTC200 system (Malvern Panalytical) at 25 °C. Proteins were purified as mentioned above. The purified proteins were loaded into the cell (20–50 µM) or syringe (400–800 µM). A total of 24 titrations were run. In each titration, 1 μl aliquot of proteins in the syringe was injected into the cell with a duration of 2 s. The time interval between 2 different titrations was 120 s to allow the titration peak to return to the baseline level. The data were analyzed with Origin7.0 from Microcal and fitted with a one-site binding model.

ITC measurement was carried out on a MicroCal iTC200 system (Malvern Panalytical, UK) at 25 °C. Both of ELMO2 and BAI1 proteins were in the buffer containing 50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT. The BAI1-EBD (~500 µM) was loaded into the syringe and injected 2 µl aliquot into the cell placed with the ELMO2-RAE (~40 µM) in each titration. The time interval was 120 s in each titration to make sure that the titration peak returned to the baseline. The experiment data were analyzed by the program of Origin7.0 (Microcal).