Table 3.
FRET analysis of the two isoforms
| NM-FRET dimer | Hsp82 dimer | Hsc82 dimer | Heterodimer |
|---|---|---|---|
| Subunit exchange rate (kse (sec−1)) | 0.034 ± 0.004 | 0.037 ± 0.006 | 0.030 ± 0.003 |
| Closing rate (kapp (min−1)) | 0.190 ± 0.010 | 0.360 ± 0.000 | 0.260 ± 0.010 |
| Change with Aha1 (percentage of kapp without co-chaperone) | 868% | 616% | |
| Change with Cpr6 (percentage of kapp without co-chaperone) | 239% | 108% | |
| Change with Cdc37 (percentage of kapp without co-chaperone) | 84% | 51% | |
| Change with Sba1 (percentage of kapp without co-chaperone) | 122% | 78% | |
| Change with Sti1 (percentage of kapp without co-chaperone) | 40% | 19% | |
| Reopening of closed state rate | |||
| Without nucleotide (t½ (min−1) | 0.564 ± 0.079 | 0.533 ± 0.085 | |
| With ATP (kapp (min−1) | 0.7 ± 0.1 | 0.7 ± 0.2 | |
| With ATPγS (kapp (min−1) | 35.5 ± 3.2 | 54.2 ± 6.0 | |
| With ATPγS and Aha1 (kapp (min−1) | 103.0 ± 2.1 | 160.0 ± 8.0 | |
| With ATPγS and Sba1 (kapp (min−1) | 47.0 ± 1.7 | 56.0 ± 2.0 | |
| With AMP-PNP (kapp (min−1) | >300 | >300 |
FRET between the NTD of one protomer and the MD of the other protomers (NM-FRET) was recorded with protomers that had fluorescent dyes attached at C61 or C385 (Hsp82) or C381 (Hsc82). FRET in the absence of nucleotides was used to obtain subunit exchange rates (kse) between protomers4. FRET in the presence of ATPγS (which stabilizes the closed state) was used to monitor the closing kinetics4. FRET chase experiments, which report on the stability of the closed complex, were performed by addition of an excess of unlabeled Hsp90 to a preformed Hsp90 FRET complex (preformed with ATPγS or AMP-PNP)4.