Figure 3

(a) Surface plasmon resonance of HtrA1 variants binding to DPMFKL-boro-V: Wild-type HtrA1 catalytic domain (green), mutant R302A (purple) and S328A (dead domain). No binding is observed for R302A or S328A. (b) Native mass-spectrometry of HtrA1 mutant R302A. Peaks with three and one green dots correspond to trimer and monomer species respectively. At high-backing pressure (panel b.A) mostly trimer species is observed (ratios for WT r = ~6/2 = ~3, R302A r = ~8/3 = ~2.7, and S328A r = ~20/5 = ~4.4) whereas increasing collision energy and/or lowering backing pressure results in an increase in monomeric species (panels b.B and b.C) and decrease of the ratio r (Δr = 0.66 WT, Δr = 0.76 R302, Δr = 0.6). Also in this sample, higher collision energy or backing pressure increase m/z ratio indicating a slight loss of native folded trimeric state. (See Supp. Info. for native MS of wild-type and S328A).