Itc200 system

The ITC experiments were performed using an ITC200 system (Micro-Cal) as previously described by Bradshaw et al.61 (link), at 25 °C in a 1X PBS buffer pH 7.4. F240 was dialyzed against this buffer overnight before the experiment. The peptides were dissolved in the final dialysis buffer. A typical experiment had F240 in the syringe (111 μM in 1X PBS pH 7.4) and gp41_583–618 or gp41_583–618 C598AC604A peptide in the cell (11 μM in 1X PBS pH 7.4). Titrations were performed at 25 °C with 17 injections of 2.42 μl aliquots, with 210–240 second intervals between injections. Heats of dilutions were measured and subtracted from each data set. Data were corrected for the heat of dilution and fitted using a nonlinear least-squares routine using a single-site binding model with Origin for ITC version 7.0383 (MicroCal). Raw data for representative experiments are included in Fig. 1B.

ITC experiments were performed at 25 °C on a MicroCal iTC200 system
(Malvern, PA, USA). hRpn13 Pru and hRpn2 (940–953) samples were dialysed
extensively against ITC buffer (20 mM sodium phosphate, 50 mM NaCl
and 10 mM βME, pH 7.0) while hRpn2 (944–953) was dissolved in
ITC buffer. One aliquot of 0.5 μl followed by 18 aliquots of
2.1 μl of 200 μM hRpn2 (940–953) or hRpn2
(944–953) were injected at 1,000 r.p.m. into the calorimeter cell
(volume 200.7 μl) that contained 20 μM or 18 μM
hRpn13 Pru, respectively. Blank experiments were performed by replacing protein
samples with buffer and the resulting data subtracted from the experimental data
during analyses. The integrated interaction heat values were normalized as a
function of protein concentration, and the data were fit with MicroCal Origin
7.0 software. Binding was assumed to be at one site to yield the binding
affinity K_a (1/K_d), stoichiometry and other
thermodynamic parameters. The peptide hRpn2 (944–953) was synthesized on a
Liberty Blue Microwave peptide synthesizer (CEM Corporation) using Fmoc
chemistry on Wang resin, cleaved from the resin and purified by high-performance
liquid chromatography (HPLC) in a manner similar to that described below for the
synthesis of FITC-hRpn2 (940–953).

ITC was performed at 25 °C using a MicroCal iTC200 system (Malvern, United Kingdom) for the protein-protein interactions. Briefly, His-TvCyP1, His-TvCyP2, and Myb3^53–180, which was previously used for structural analysis of Myb3^{35 (link)}, were purified, and kept in 20 mM phosphate buffer at pH 6.0 containing 50 mM NaCl. Two microliters of 1.5 mM Myb3^53–180 was injected at intervals into 300 μl of 150 μM His-TvCyP2 or His-TvCyP1 in ITC cell. The data were collected and analyzed using the software Origin 7.0. The binding isotherms were fitted to the one-site binding model, giving values of the stoichiometry (n) of the interaction, the enthalpy of binding (ΔH) and the association constant (K_a), which is used to derive the binding affinity dissociation constant (K_D).

ITC experiments were performed using the MicroCal iTC₂₀₀ system at 25°C as described previously (17 (link)). In brief, amino acid ligands were dissolved in ITC buffer at a concentration of 500 to 1,000 μM. SBD proteins were concentrated to 30 to 100 μM using a Millipore 3-kDa-molecular-weight-cutoff (MWCO) filter (Millipore, USA). The ligands in a syringe were added stepwise to SBD proteins in an ITC cell with a stirring speed of 1,000 rpm. Heat transfer (microcalories per second) measured from injections was analyzed to determine the dissociation constant (K_D) using nonlinear regression fitting of a single-binding-site model (MicroCal ORIGIN software, OriginLab, USA).