Isothermal Titration Calorimetry and Data Analysis

The protein samples were dialyzed against buffers containing 20 mM HEPES pH 7.5 and 100 mM NaCl, or 20 mM Sodium citrate pH 5.0 and 100 mM NaCl. A MicroCal ITC-200 isothermal calorimeter was used to carry out calorimetric experiments at 20°C with stirring at 750 rpm (Malvern Panalytical, UK). The interface between the cell, containing 15-30 μM eE2, and the syringe was equilibrated with 0.4 μL (0.14–0.19 μM tCD81-LEL and 0.30–0.58 μM hCD81-LEL) of ligand with a spacing of 0.8 seconds, followed by 16 subsequent injections of 2.45 μL (0.87–1.17 μM tCD81-LEL and 1.82–3.51 μM hCD81-LEL) with a spacing of 180 seconds.

ITC thermograms were integrated using the NITPIC program⁴⁰ and normalized peak area plots were fitted using SEDPHAT⁴¹. The A + B ⇄ AB heterodimer model was used to determine eE2-CD81 interaction. Enthalpogram fitting parameters included K_D and ΔH while eE2 was considered the incompetent fraction. Binding parameter confidence level of 95% and error surface of the fit were estimated with SEDPHAT. ITC measurements were performed at the NHLBI Biophysics Core Facility.