Origin v7

Heat flow resulting from binding of lipid small unilamellar vesicle (SUV's), to NAcPLB₂₃WT or NAcPLB₂₃R14Δ was measured using the ultrasensitive iTC₂₀₀ MicroCalorimeter (MicroCal LLC, Northampton, MA). Reaction cell volume and total injection volume were 200 µl and 40 µl respectively. Experiments were performed at 25°C, at a power reference setting of 6 µcal/sec with stirring at 800 rpm. Data analysis was carried out using the Origin v.7 software (MicroCal) and fitted using the ‘one set of sites’ model. The reaction cell contained a 100 µM solution of peptide in 10 mM Tris; 1 mM EDTA, 30 mM NaCl; pH 7.4. Lipid SUV's comprising 10 mM 2∶1 L-α-dimyristoylphosphatidylcholine/L-α-phosphattidylglycerol (DMPC/DOPG) in the same buffer, were prepared using a probe sonicator and sonicated at 50% duty cycle for 2×30 s and injected via the syringe. Titrations were carried out at intervals up to 2 minutes in 1 or 2 µl aliquots following an initial 0.5 µl discard aliquot. Each injection generates a heat of reaction, determined by integration of the individual peaks from the heat flow trace.

Isothermal titration calorimetry (ITC) was performed using the MicroCal VP-ITC titration calorimeter equilibrated to 25°C, essentially as previously described (15 (link), 26 (link), 46 (link)). The proteins (20 to 100 μM) were placed in the sample cell, and the syringe was loaded with 2.5 mg/ml polysaccharide or 0.5 to 2 mM oligosaccharide. Following an initial injection of 2 μl, 25 subsequent injections of 10 μl were performed with stirring at 280 rpm, and the resulting heat of the reaction was recorded. Integrated heats were fit to a single-site model using Microcal Origin v7.0 to derive n, K_a, and ΔH values.

ITC experiments were performed using an ITC200 instrument (MicroCal, Wolverton Mill, UK) at 24°C. SmN constructs (168–196, 168–240) and an SmN peptide (219–229) at 1 mM, 1 mM and 5 mM, respectively, were titrated into OCRE (100 μM, 100 μM and 1 mM, respectively). For the mutation analysis, purified OCRE mutants (2 mM) were titrated to wild type SmN (29.5 μM) or SmB (40 mM). All purified proteins were in the same buffer, 20 mM sodium phosphate pH 6.5, 50 mM NaCl. The lyophilized peptide was dialyzed against water, lyophilized again, and then dissolved in the same buffer as the protein. The heat of dilution was measured by titrating SmN or OCRE mutants into buffer. The titration protocol consisted of one initial injection of 0.4 μL followed by 38 injections of 1 μl of the ligand into the protein sample with intervals of 120 s, allowing the titration peak to reach the baseline. Data were calculated using the program Origin v7.0 (MicroCal) and duplicates were measured for all the experiments.