Fig. 8: CspZ-YA_C187S and CspZ-YA_I183Y maintained the recognition by protective CspZ IgGs at higher temperatures for a longer time.

A Untagged or histidine-tagged CspZ-YA or mutant proteins (10 µM) in PBS buffer were subjected to the thermal shift assays with the emission wavelength of 623 nm, as described in the materials and methods section. Shown are the fluorescence intensities of each of the CspZ-YA proteins under temperatures ranging from 25 to 99 °C from one representative experiment. The melting temperature (Tm) was extrapolated from the maximal positive derivative values of the fluorescence intensity (d(RFU)/dT) and shown in Table S3. B, C One µg indicated CspZ-YA proteins was incubated at 4 or 37 °C for 6- or 24-h prior to being coated on microtiter plate wells. The microtiter plate wells immobilized with each of these proteins before incubation (0-h) were included as unincubated control. The ability of the CspZ monoclonal IgG, B 1139c or C 1193c, to recognize each of these CspZ-YA proteins were determined using ELISA in the section “Accelerated stability study” in Materials and Methods. The work was performed on four independent experiments (one replicate per experiment). Data are expressed as the percent binding, derived by normalizing the levels of bound 1139c or 1193c from the wells coated with each of the CspZ-YA proteins in different incubating conditions to that in the unincubated control. Data shown are the mean ± standard deviation of the percent binding of 1139c or 1193c from n = 4 experiments. Statistical significance (p < 0.05, Kruskal–Wallis test with the two-stage step-up method of Benjamini, Krieger, and Yekutieli) of differences in percent binding between groups are indicated (“#”). B CspZ-YA 0 h: CspZ-YA 37 °C, 24 h p = 0.0019. His-CspZ-YA 0 h: His-CspZ-YA 37 °C, 24 h p = 0.0484. C CspZ-YA 0 h: CspZ-YA 37 °C, 24 h p = 0.0003. His-CspZ-YA 0 h: His-CspZ-YA 37 °C, 24 h p = 0.0028. Source data are provided as a Source Data file.