Figure 3.

Interactions of the β clamp with the δ subunit of DnaX. (A) Representative SPR results analyzing interaction of δ (4 μM) with various β clamp proteins (∼100 RU). Clamps examined include the his₆-tagged β⁺ homodimer (β⁺), the his₆-tagged β^C homodimer (β^C), the his₆-tagged β⁺/myc-tagged β⁺ heterodimer (β⁺/β⁺) and the his₆-tagged β⁺/myc-tagged β^C heterodimer (β⁺/β^C). Theoretical R_max values for binding of a single δ subunit (δ1), or for two δ subunits (δ2) are indicated by dashed lines. (B) Titration of δ with his₆-tagged β⁺. Increasing concentrations of purified δ (0.1, 1, 10, 25, 50, 100, 250, 500, 750 and 1000 nM) were injected over ∼100 RU of his₆-tagged β⁺ captured on the anti-Penta•His chip surface. (C) Comparison of sensorgrams from low (25 nM) and high (1 µM) concentrations of δ injected over ∼100 RU of his₆-tagged β⁺ captured on the anti-Penta•His chip surface from panel (B) highlighting differences in association (k_a) and dissociation rates (k_d). The amplitudes (R_max) of these SPR traces were normalized to each other to facilitate a direct comparison of the association and dissociation phases of the curves. (D) Titration of δ with β⁺/β^C. Increasing concentrations of purified δ (10, 25, 50, 100 and 500 nM) were injected over ∼100 RU of β⁺/β^C captured on the anti-Penta•His chip surface. (E) The double-injection experiment was performed as outlined in the cartoon (bottom). ∼100 RU of his₆-tagged β⁺ was captured on the anti-Penta•His chip surface (injection 1). Saturating levels (10 µM) of δ were injected (injection 2), forming the β–δ₂ complex (R_max approaching 100 RU). Flow was switched to buffer, allowing for the more loosely bound δ (δ2) to dissociate, resulting in a β–δ₁ complex. Increasing concentrations of δ (0, 1, 10, 25, 50, 100, 250, 500, 750 and 1000 nM) were then injected over the β–δ₁ complex (injection 3) to determine the binding affinity for the second δ (δ2). (F) Summary table of kinetic constants for various clamp–δ interactions derived from SPR results summarized in (A–E). Results shown for β⁺–δ were determined using the his₆-tagged β⁺ homodimer, and were indistinguishable from those observed with β⁺/β⁺. An interaction of β^C with δ was not detected (nd).