Figure 5. pH-dependencies of YscU_C dissociation in vitro and Yop/YscU_CC secretion in vivo.

(A) Thermal up- and down-scans of YscU_C at pH 6.0, monitored with CD spectroscopy at 220 nm in the absence of calcium. The thermal signature of YscU_C displayed one large-amplitude transition at 55°C. Up- and down scans of YscU_C are shown in red and blue circles, respectively. (B) The pH-dependency of the YscU_C monitored with CD spectroscopy at 220 nm. (C) Chemical shift perturbations of YscU_C quantified from ¹H-¹⁵N HSQC spectra, in response to a pH-shift from 7.4 to 6.0, displayed against the primary sequence. The blue line indicates the threshold value (0.05 ppm) used in Figure 5D. The chemical shift perturbation of the two histidines at positions 266 and 324 are shown in red. (D) Structural distributions of residues that show significant chemical shift perturbations in response to a pH-shift from 7.4 to 6.0 are shown in red on the YscU_C structure (2JLI.PDB). The YscU_CN and YscU_CC fragments are colored orange and gray, respectively. The two histidine residues (266 and 324) in the folded part of YscU_C are indicated. (E) Coomassie stained gels show Yop secretion under different pH conditions. (top panel) “pellet” indicates intracellular proteins; (middle panel) “supernatant” denotes secreted proteins. The YopJ protein (black box) was subjected to densitometric analysis for quantification of secretion levels (see Table 2). (bottom panel) The pH-dependency of YscU_CC secretion was visualized on immunoblots with anti-YscU_CC peptide antibodies.