Supplementary Figure 8: Fluorescence-based assays for Rpn11 ubiquitin binding and cleavage.

(a) Example time-based polarization measurements of the cleavage of 5 and 10 μM Ub-Lys-TAMRA by 1.25 μM Rpn11-Rpn8. (b) Example single-turnover kinetics measurement for the cleavage of 100 nM Ub-Lys-TAMRA by 450 μM Rpn11-Rpn8. The data are fit by a single exponential, with a calculated k_cat = 0.95 min⁻¹. (c) Michaelis-Menten curve for Ub-Lys-TAMRA cleavage by Rpn11, where k_cat was constrained to the experimentally determined value in (b). Limitations in substrate solubility precluded using Ub-Lys-TAMRA at concentrations higher than K_M, so measurement of a complete curve was not possible. The estimated K_M for Ub-Lys-TAMRA cleavage by Rpn11 is 20 μM. (d) Tryptophan fluorescence-based assay of K48-linked di-ubiquitin binding to Rpn11^V80A. Tryptophan fluorescence of 5 μM Rpn11-Rpn8 heterodimers was measured in the presence of Lys48-linked di-ubiquitin at concentrations between 0 and 500 μM, as discussed in the methods. Triplicate fluorescence measurements were averaged and fit to a simple binding curve with a K_D of 67 μM.