Figure 4

Oligomeric Structure and the Role of Disulfide Bonds. The dashed black line indicates the elution profile of the column calibration protein mix A (left to right: ferritin, conalbumin, carbonic anhydrase and ribonuclease A). The MaMsvR monomer is 29.2 kDa. (a) The elution profile for non-reduced MaMsvR (0.65 mg loaded) is indicated by the solid black chromatogram trace. Inset is an SDS-PAGE of MaMsvR fractions collected during the gel filtration run (a-f). (b) The elution profile for reduced (0.84 mg with 2 mM β-ME in the elution buffer) MaMsvR is indicated by the solid black chromatogram trace. Inset is an SDS-PAGE of MaMsvR fractions collected during the gel filtration run (a-d). (c) Immunoblot of an SDS –PAGE gel probed with a Strep-tag antibody where MaMsvR was prepared and subjected to electrophoresis (1 pmol each protein) in non-reducing SDS-PAGE sample buffer (N) and reducing (R) SDS-PAGE sample buffer on a 15% Tris-Glycine gel (no SDS). A reduced and boiled sample of MaMsvR is shown as a control (RB). The monomer is designated by M, whereas D and T indicate bands corresponding to a possible dimer and tetramer, respectively. (d) EMSA performed with Ma P _msvR and native MaMsvR and three C to A variants of MaMsvR. The control DNA only lane is indicated by a (-). The (+) lanes contain the indicated MaMsvR variant in the absence of any reducing agent. The (R) lanes contain the indicated MaMsvR variant and 5 mM DTT as a reducing agent.