Figure 2. Proteasome-substrate interaction kinetics by the SM method.

A. Schematics showing the experimental design, where purified 26S proteasome was immobilized on passivated coverslip using anti-20S antibody. Ubiquitin was fluorescently labeled and conjugated to substrates in solution. Sample pictures capture ubiquitin signals on the surface with either 26S proteasome (+26S) or antibody only (no 26S). B. The average dwell time on the proteasome for different substrates (or free ub-chain) with X-number of conjugated ubiquitin, measured by the SM method. The distributions of individual dwell times are shown in Fig. S15. Errorbars represent standard deviation of the mean. ‘*’: data for Ub-chain from 6~9 is not shown due to insufficient events. C. Cooperative and stochastic mechanisms affect binding enthalpy and entropy respectively. By each mechanism, the expected relationship between dwell time t_b and the number of ubiquitins N is shown below. D. Dwell time on the proteasome(right), or its logarithm(left), for securin and cyclinB vs. the number of conjugated ubiquitins. The red line shows a linear fitting. The ratio of p-values by fitting the red segment (in linear scale) with either a linear model (p_l) or an exponential model (p_e) is shown. Δg_ub is the binding free energy per ubiquitin on the proteasome, calculated from the slope of the binding curve.