FIGURE 1.

Biochemical characterization of Pa DpsL in solution. (A) The oligomeric state of Pa DpsL was assessed with the aid of a calibrated size exclusion column. Pa DpsL in 50 mM Tris pH 7.5 and 1 mM MgSO₄ elutes as a 12-mer (black trace). Removal of the MgSO₄ from the buffer in the sample and from the chromatography buffer causes the disassembly of the 12-mer (blue trace). Addition of 1 mM MgSO₄ to the protein eluting in the blue trace chromatogram and to the chromatography buffer does not enable the full reassembly of 12-mer (red trace). (B) SDS PAGE shows that incubating Pa DpsL in the presence of MgSO₄ stabilizes Pa DpsL; in the absence of MgSO₄ the protein is proteolytically cleaved and degraded. (C) In gel digestion and LC-MS/MS analysis of Pa DpsL. SDS PAGE lane (A) 15 μM Pa DpsL in 50 mM Tris (pH 7.5) + 1 mM MgSO₄. Lane (B) 15 μM Pa DpsL incubated for 6 h in 50 mM Tris (pH 7.5). Lane M: molecular weight marker. Bands 1, 2 and 3 were excised, the protein digested with trypsin, and the peptides analyzed by LC-MS/MS. All the expected peptides were detected in bands 1 and 2, indicating that the protein is intact, whereas the residues shown in blue were not detected in band 3, indicating that proteolytic cleavage starts at the amino terminus. (D) Incubating Pa DpsL in 50 mM Tris with MgSO₄, MgCl₂, or Na₂SO₄ for 6 h prior to SDS PAGE shows that both Mg²⁺ and SO₄ ^2- are required to prevent proteolytic cleavage.