Thermal Stability of Gαi Proteins

Gαi proteins were exchanged in CD buffer (10 mM K₂HPO₄, 500 μM MgSO₄, 500 μM tris(2-carboxyethyl)phosphine (TCEP)) using an Amicon centrifugal filter unit (MilliporeSigma, #UFC901024) at 3800 × g, diluted to 15 μM (pH 6.0, 6.4, 6.8, 7.2, or 7.6) and centrifuged (14,000 rpm) for 10 mins at 4 °C. Temperature dependent CD experiments were performed on a Jasco J815CD spectrometer using 15 μM WT or Gαi variants protein in a 1 mm path-length quartz cuvette (Hellma Analytics). Thermal melts were obtained at 222 nm, over a temperature range of 20–95°C, using a temperature increment of 1 C/min. The CD spectral scans were collected for Gαi proteins at different fixed temperatures (e.g. 45°C, 55°C and 65°C) by taking CD measurements every 1 nm from 200–250 nm. Secondary structure was evaluated using the online server BeStSel (Beta Structure Selection)^{9 (link)}.