Figure 5. Sedimentation velocity analysis of KdpE_DBD—kdpFABC_BS association.

A. Continuous distribution of sedimentation coefficients [c(s)] as a function of increasing concentration of protein against a fixed concentration of kdpFABC_BS DNA (0.5 µM). The protein concentrations used varied between 0.25 and 16 µM as shown. The largest complex with sedimentation coefficient of 4.1 S was observed at protein concentration of 4 to 16 µM. Independent experiments established the sedimentation coefficients of KdpE_DBD and kdpFABC_BS at 1.4 S and 2.8 S respectively (data not shown). B. A plot of the weight average sedimentation coefficients (S_w) against the concentration of KdpE_DBD is shown. Analysis of the isotherm indicated that DNA was saturated beginning at 8-fold molar excess of KdpE_DBD protein. C. SV c(s) distributions comparing binding of KdpE_DBD to the S1 and S2 sites individually and to the both sites simultaneously. Wild-type DNA with both sites intact (kdpFABC_BS), functional S1 (kdpFABC_BS —7) and S2 (kdpFABC_BS —1) sites were analyzed with a 16-fold molar excess of Kdp_DBD. Complexes with DNA possessing single sites have sedimentation coefficients of 3.5 S whereas when both sites were occupied a 4.1 S species was formed.