*4.3. EPR Spectroscopy*

For spin-label EPR measurements, 5 μL of 5-SASL or 16-SASL spin label solution (Sigma-Aldrich) in ethanol (2 mg/mL) was added to 1 mg of lipid in chloroform solution (resulting in a ca. 50:1 lipid/spin label molar ratio). After vortexing, the solution was dried under nitrogen gas and incubated under vacuum overnight at room temperature. The dried samples were hydrated with PBS buffer (pH 7) above the melting temperature of the lipid. HSPB1 protein was added to the lipid suspension from an 11 mg/mL stock solution in PBS, resulting in a lipid:protein molar ratio of 100:1 in the samples.

A glass capillary with an internal diameter of 1 mm was then filled with 10 μL of spin-labeled sample. The EPR spectra were recorded using either a Bruker (Rheinstetten, Germany) ECS-106 or a Bruker ELEXSYS-II E580 X-band spectrometer at room temperature, with the following instrument settings: microwave frequency of 9.4 GHz, microwave power of 5 mW, field modulation of 0.7 G, scan range of 100 G, and conversion time of 40.96 s. Final spectra were the mean of four scans. Data analysis was performed using Igor Pro (Wavemetrics, Inc.; Portland, OR, USA).
