Supplementary Figure 10: Covalent circularization stabilizes MSPs against digestion by V8 protease without and with lipids.

(a, b) SDS-PAGE analysis of the proteolysis of lipid-free MSPΔH5 and cNW9. Samples were treated with V8 protease for 0 min (before addition of V8), and for 1, 3 and 18 hours. Lanes are labeled with times of protease treatment. Proteolysis was performed in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl at 37°C and using a protein:protease ratio (w/w) of 1000:1. Treatment of MSPΔH5 with V8 resulted in the appearance of a large peptide, which is close in size to MSPΔH5 and is not discernible until about 18 hours after addition of V8; this peptide may be generated earlier but is not visible due to the low amount or overlap with uncleaved MSPΔH5. The intensity of the MSPΔH5 band decreased by 75% after 3 hours and by 93% after 18 hours. On the other hand, the intensity of the cNW9 band decreased by only 20% after 18 hours. A band that corresponds to linearized NW9 was observed after 3 hours and increased in intensity after 18 hours. (c, d) SDS-PAGE analysis of the proteolysis of nanodiscs assembled with MSPΔH5 and cNW9. Samples were treated with V8 protease for 0 min (before addition of V8), and for 20 min, 1, 3 and 18 hours. Proteolysis was performed at 37°C at pH 7.5 in 20 mM Tris-HCl, 100 mM NaCl using a 100:1 protein:protease (w/w). The band intensity of MSPΔH5 decreased by 81% after 3 hours. There was no decrease in the cNW9 band intensity up to 3 hours. The ImageJ software was used for analyzing band intensities.