FIG 3.

Determination of stoichiometry for the ExbB₄-ExbD₁-TonB₁ complex. (A) Protein and detergent contributions of the DDM-solubilized ExbB-ExbD-TonB complex were measured by SEC-MALLS using the three-detector method. From candidate extinction coefficients, the best derived stoichiometry was ExbB₄-ExbD₁-TonB₁. The resultant complex size was ∼230 kDa, ∼40.5% of which is DDM. (B) Native agarose gel electrophoresis (pH 7.5) separated proteins based on pI. Lysozyme (pI 11.0) migrated toward the cathode (indicated by a minus sign); BSA (pI 5.1) migrated toward the anode (indicated by a plus sign). The three-protein complex showed less migration than ExbB₄-ExbD₂, consistent with its predicted electronegativity. For clarity, the brightness of the image has been adjusted.