Table 1.

Thermal stability of the Hsp90 isoforms and mechanical stability of their individual domains

Isoform	Tm (°C)	Domain	Expected length (nm)	Measured length (nm)	Unfolding force 1 (pN)	Unfolding force 2 (pN)	Unfolding force 3 (pN)
Hsp82	60.4 ± 0.5	Hsp82 NTD	71.8	69.5 ± 2.5	15.5 ± 1.6	12.1 ± 1.3	16.3 ± 2.2
Hsp82 + ATPγS	63.3 ± 0.4	Hsp82 MD	84.5	85.0 ± 1.5	19.2 ± 1.6
Hsc82	57.1 ± 0.2	Hsp82 CTD	40.5	40.2 ± 1.5	11.1 ± 2.0
Hsc82 + ATPγS	60.8 ± 0.4	Hsc82 NTD	71.8	66.8 ± 1.7	15.0 ± 1.9	10.2 ± 0.88	15.1 ± 2.6
		Hsc82 MD	84.5	83.9 ± 1.5	20.1 ± 1.8
		Hsc82 CTD	40.5	42.3 ± 2.0	10.8 ± 1.9

Melting temperatures (T_m) of the Hsp90 isoforms were determined by tracking SYPRO orange binding upon thermal unfolding in the absence or presence of ATPγS. Means of three technical replicates and standard derivation are shown. To determine the mechanical stability of the Hsp90 domains of the isoforms optical trapping experiments were performed at 30 °C and the expected gains in contour length were compared with the measured gains in contour length. The indicated domains were pulled at different speeds and the average unfolding forces are indicated: Unfolding force 1: average unfolding force at 500 nm/s, unfolding force 2: average unfolding force at 20 nm/s, unfolding force 3: average unfolding force at 20 nm/s in the presence of 10 µM RD. Average forces and SD were calculated from sample sizes of: 426 events and 132 events for unfolding force 1 for Hsp82 and Hsc82, respectively; 82 events and 38 events for unfolding force 2 for Hsp82 and Hsc82, respectively; and 16 events and 34 unfolding events for unfolding force 3 for Hsp82 and Hsc82, respectively.