Figure 5: The Hsp90/p23 system is directed and coupled to ATP hydrolysis.

(a) Schematics of the four-colour FRET measurement with a twofold labelled Hsp90 heterodimer (Atto488, Atto550) and labelled p23 (Atto594) encapsulated in vesicles with a radius of around 100 nm, which contain α-hemolysine pores that allow entering of labelled ATP (Atto647N, 200 nM). (b) Example curves showing different types of transitions as depicted by the colour code. (c) Approximate values for the relative frequency (probability) of the observed transitions. Exact values and confidence intervals are given in Table 1. (d) Relative energy of the different states estimated from the transition probabilities (Table 1) and the occupancy of the states (Supplementary Fig. 7), formula see Supplementary Notes, page 18. The state after hydrolysis is called state 3 (the sum of all p23 bound states), as we cannot tell at what stage the energy of hydrolysis is utilized (hydrolysis, Pi or ADP release). Estimates: ΔG_1->2=1.1 k_BT; ΔG_2–>3a=−2.4 k_BT; ΔG_3a–>3=4.9 k_BT; ΔG_3–>1=−3.6 k_BT.