FIGURE 2.

Binding kinetics to Rpn1 or Rpn2. Rpn1 and Rpn2 were immobilized on separate channels (1700 and 1100 response units (RU)) of a ProteOn surface plasmon resonance sensor and injected with increasing concentrations of their potential binding partners followed by buffer wash. Positive associations are displayed for Rpn1-Rad23 (A), Rpn1-Dsk2 (B), Rpn1-Ubp6 (C), and Rpn2-Rpn13 (D). The response data (dashed lines) for association and dissociation are shown for a series of concentrations (arrows) overlaid with a model fit (solid line). Sensograms in A, B, and D were fit to the Langmuir model for 1:1 binding stoichiometry. Sensograms in C (Rpn1-Ubp6 response) were fit to an HLPR, assuming two-binding sites. Association (k_on) and dissociation (k_off) rate constants derived from a global fit of primary response data over the entire concentration range for each pair to the corresponding model are summarized in Table 1. E, binding isotherms for Ubp6^UBL (circles), Rad23^UBL (triangles), Dsk2^UBL (diamonds), Ddi1^UBL (×), and ubiquitin (squares) derived from equilibrium SPR measurements. Normalized equilibrium response is plotted as a function of soluble protein concentration; the fitting curves correspond to dissociation constants shown in Table 2.