Fig. 4: Nucleotide dependent conformations and their dynamics for yHsp90 and hHsp90 measured by smFRET.
From: Evolution of the conformational dynamics of the molecular chaperone Hsp90
A SmFRET efficiency histograms of yHsp90 in the apo state (black), in the presence of AMP-PNP (green) and in the presence of ATPγS (orange). B SmFRET efficiency histograms of hHsp90 in the apo state (black), in the presence of AMP-PNP (green) and in the presence of ATPγS (orange). FRET histograms are of representatives from at least two independent measurements unless otherwise mentioned. C–F 2D histograms of FRET efficiency vs. donor fluorescence lifetime in the presence of an acceptor (τD(A)) for C yHsp90 w/AMP-PNP, D yHsp90 w/ATPγS, E hHsp90 w/AMP-PNP and F hHsp90 w/ATPγS. Black lines indicate the static FRET line whereas both the red and blue curved lines depict dynamic FRET lines. G Accessible volume (AV) calculations of residues C61 for yHsp90 (left panel) and residue C70 for hHsp90 (right panel) labeled with Atto532 as a donor and Atto643 as an acceptor dye. Distances of 88.8 Å (AV) versus 89.6 Å (measured) for yHsp90 and 89.9 Å (AV) and 82–86 Å (measured) for hHsp90 were determined. H, I Dynamic photon distribution analysis (PDA) for yHsp90 and hHsp90, respectively, in the presence of ATPγS. The open conformation is shown in violet, the closed conformation in blue, a compact conformation with high FRET efficiency in red and the contribution of dynamically interconverting populations are shown in yellow. For experiments with yHsp90, nucleotides were preincubated with the protein for 2 h. In the case of hHsp90, the preincubation time was 4 h.
