Nano itc isothermal titration calorimeter

Binding assays of MjCobT and NaMN substrate were performed using a Nano ITC isothermal titration calorimeter (TA instruments). MjCobT was dialyzed against Tris-HCl buffer (25 mM, pH 7.5) containing NaCl (150 mM); NaMN was solubilized in the final protein dialysate. Samples were degassed for 20 min at 25°C before use. MjCobT protein was present in the sample cell at 33 μM under constant stirring at 350 rpm, and NaMN was present in the injection syringe at 1 mM. Every 5 min, injections (2.4 μL) were made into the sample cell and heat released was recorded. Experiments were conducted at 25°C. Data were analyzed using NanoAnalyze software (TA instruments).

The interaction of NBD of human mortalin with modified ADP analogues was
measured by isothermal titration calorimetry (ITC) using a Nano ITC isothermal
titration calorimeter (TA Instruments) at 25 °C. A library of
commercially available ADP analogues with varied substituents at the N6- or
2-position were purchased (Biolog). Concentrations were determined using a 660
nm Protein Assay (Pierce). Nucleotide free mortalin-NBD and modified ADP
analogues, at 50 μM and 500 μM respectively, were prepared in
binding buffer containing 20 mM HEPES/NaOH (pH 7.8), 50 mM NaCl, 5 mM KCl, 5 mM
MgCl₂, and 5 mM Na₂HPO₄. The enthalpy
change for each injection was calculated by integrating the area under the peaks
of the recorded time course of the power change. The data were analyzed with
NanoAnalyze v3.8.0 software using the nonlinear least-squares fitting to a
single-site binding model to calculate the dissociation constants
(K_d), binding stoichiometry (n), the binding enthalpy change
(ΔH), and binding entropy change (ΔS).