Figure 2.

Dissociation of the Hsp90 dimer. (A) The confocal laser foci are placed at different positions in the measurement chamber representing different times after dilution. The design of the measurement channel allows us to measure dissociation rates between 5 ms 100 s (B) Stoichiometry histograms are shown for two different measurement positions, i.e. dilution times. The corrected dimer fraction at each time point is calculated from the acceptor-only (S < 0.2) and the FRET population (0.2 < S < 0.8) using equation (14). A FRET efficiency histogram is shown for a time point of 1 s after dilution indicating that Hsp90 is mainly populated in an N-terminally open conformation. (C) The corrected dimer fractions for two measurement series, namely 1 μM Hsp90 APO (black circles) and 1 μM Hsp90 + 2mM ATP (red triangles), are plotted on a logarithmic timescale. The data is fitted with two- or three-exponential decays. Optimal fits result in two dissociation rates for Hsp90 APO, k_dis,APO,high=52 s^-1 with amplitude A_APO,high=32 % and k_dis,APO,low=0.025 s^-1 with A_APO,low=68 %, and three dissociation rates for Hsp90+ATP, k_dis,ATP,high=51 s^-1 with A_ATP,high=28 %, k_dis,ATP,mid=0.09 s^-1 with A_ATP,mid=36 %, and k_dis,ATP,low=0.006 s^-1 with A_ATP,low=36 %. The square marks represent average dimer fractions for different Hsp90 APO concentrations in the incubation chamber. This enables determination of the equilibrium dissociation constant K_D and the association rate k_ass. The uncertainties are the standard deviation of three separate measurements.