Flow Chart of FSEC-TS The protein sample, after heat treatment and centrifugation, is loaded onto a SEC column, connected to a fluorescence detector to monitor GFP or tryptophan fluorescence. The panel labeled ‘‘Fluorescence’’ shows a hypothetical elution profile from GFP or tryptophan fluorescence.

Thermostability Profiles of zfP2X4 and GluCl (A, C, and E) Representative FSEC profiles from EGFP (A), EGFP-ΔP2X4-A (C), and GluClcryst (E), heated at the respective temperatures, detected by GFP fluorescence (A and C) or by Trp fluorescence (E). (B, D, and F) Representative melting curves of EGFP (B), EGFP-tagged and EGFP-free ΔP2X4-A (D), and GluClcryst and GluCl-EGFP (F). Melting temperatures (Tm) were determined by fitting the curves to a sigmoidal dose-response equation. The fitted curves are shown as a black line. See also Figure S1.

Effects of Additives on the Thermostability of zfP2X4 (A) The normalized peak heights from EGFP-fusion ΔP2X4-A, heat-treated at 50°C for 10 min, in the presence of the respective additives. The peak heights were normalized to that from the sample at 4°C without any additive. (B–D) Chemical structures of novel cholesterol derivatives, hexaethyleneglycol hydroxyethyl cholesterol (B) maltoside hydroxyethyl cholesterol (C), and phosphocholine hydroxyethyl cholesterol (D), employed in the FSEC-TS studies are shown. Structures were created using the ChemDraw (Cambridge Scientific Computing). See also Figure S2 and Table S1.

Effects of Lipids on the Thermostability of GluCl The normalized peak heights from GluClcryst heat-treated at 57°C for 10 min, in the presence of the respective lipids. The peak heights were normalized to that from the sample at 4°C without any additive. See also Figure S3 and Table S2.