Oligomeric state determination

We characterized the oligomeric state of purified human CST (hCST) as it was initially one of our most promising constructs but did not grow well-diffracting crystals. Compared to mCST, it has an identical migration time on a gel filtration column (data not shown); therefore, we expect that the results from the following experiments will also be applicable to mCST. The molecular weight of the protein component of the purified hCST-lipid-detergent complex, which has a theoretical molecular weight of 37 kDa, was determined with the ‘SEC-UV/LS/RI’ approach using the ‘three detector’ method (Arakawa et al., 1992; Folta-Stogniew, 2006; Hayashi et al., 1989). 250 μl of purified hCST at 0.8 mg/ml was loaded onto a Superdex 200 column equilibrated in Buffer B. The output from the column was then passed through UV absorbance, light scattering, and refractive index detectors. The chromatography and analysis of the data were performed at the Biophysics Resource of Keck Facility at Yale University.

For the cross-linking reactions, either disuccinimidyl suberate (DSS; Thermo Fisher Scientific) or bis(sulfosuccinimidyl)suberate (BS³; Thermo Fisher Scientific) were added at the indicated concentrations to 1 mg/ml purified hCST in Buffer A. The reactions were incubated for 30 min at room temperature (RT) and then quenched by adding 1 M Tris-HCl pH 7.5 to a final concentration of 100 mM. Samples were then incubated for 15 min at RT before being run on a 12% SDS-PAGE gel.