1. Cooperative Nucleotide Binding in Hsp90 and Its Regulation by Aha1
Philipp Wortmann, Markus Götz, Thorsten Hugel 
Biophysical Journal 
Volume 113, Issue 8, Pages (October 2017)
DOI: /j.bpj
Copyright © 2017 Biophysical Society Terms and Conditions

2. Figure 1
Data acquisition, data analysis, and state allocation. (a) Pictogram of the studied system consisting of an Hsp90 dimer with the labels Atto488 (blue) and Atto550 (green) attached and the reporter nucleotide AMP-PNP∗ in solution, labeled with Atto647N (red). The protein is immobilized by NeutrAvidin/biotin interaction on the top of the flow chamber and excited by an evanescent field using alternating laser excitation with a blue and a green laser in a prism-type total internal reflection fluorescence geometry. The fluorescence light is collected by an objective, separated by dichroic mirrors, and detected with electron-multiplying charge-coupled device cameras. (b) Fluorescence intensity traces of a single particle after the excitation of the blue (top) and the green (center) dye measured in the absence of additional unlabeled nucleotide or co-chaperone. The partial fluorescence traces (PFemex, with excitation of the ex dye and emission of the em dye) calculated from the intensity traces are shown below. (c) Pictograms of the distinguishable states and the respective identifiers used in this work. The first two populations represent the same functional state, namely open Hsp90 with nucleotide bound. (d) 3D representation of the Gaussians (isosurface at FWHM) fitted to the partial fluorescence data, which represent the five different populations. The same color code as in (c) is used. (e) The resulting state allocation for the fluorescence intensity traces is shown in (b).
Biophysical Journal  , DOI: ( /j.bpj )
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3. Figure 2
The average dwell time of the reporter nucleotide AMP-PNP∗ bound to Hsp90 is prolonged by additional nucleotide. (a) Pictogram of the observed dissociation of labeled AMP-PNP∗ (PNP) from the Hsp90 dimer. (b) Average dwell time of AMP-PNP∗ bound to Hsp90 in the absence of additional nucleotide (PNP∗) and in the presence of 250 μM unlabeled ATP or unlabeled AMP-PNP. The underlying dwell time distributions are shown in Fig. S9. Error bars represent the SD estimated from jackknife resampling. Differences between the dwell time distributions are significant with ∗∗∗p < To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

4. Figure 3
The effects of nucleotide on Hsp90’s conformation and its state transitions. (a) Populations of open and closed conformation for Hsp90 bound to AMP-PNP∗ (normalized to unity) in the absence of additional nucleotide (PNP∗) and in the presence of 250 μM ATP or AMP-PNP. Error bars represent the SD within 10 subsets, each comprising 75% of the full dataset. The addition of nucleotide results in a significant population shift with ∗∗∗p < (b) Transition rates for Hsp90 bound to labeled AMP-PNP∗ in dependence on additional nucleotide. Error bars represent the 99% CI; differences with ∗∗p < 0.01 (CIs do not overlap) are highlighted. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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5. Figure 4
The effects of Aha1 (10 μM), ATP (250 μM), and Aha1 combined with ATP on Hsp90 and AMP-PNP∗. (a) The mean dwell time of AMP-PNP∗ on Hsp90 is significantly (∗∗∗p < 0.001) increased by Aha1. The effect observed for Aha1+ATP is smaller than for Aha1 or ATP alone. Error bars are calculated as in Fig. 2. (b) Effects on the normalized population of open and closed conformation for Hsp90 bound to AMP-PNP∗. Error bars are calculated as in Fig. 3 a. (c) Transition rates and the effects of nucleotide, co-chaperone, and their combination. Errors bars represent the 99% CI; differences with ∗∗p < 0.01 are highlighted. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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6. Figure 5
The effects of ATP and Aha1 on the state transitions of Hsp90 and their possible structural origins. (a) A minimal model for the state transitions of Hsp90 in the presence of AMP-PNP∗ and the effects of ATP and Aha1 on these transitions. Only the most frequent transitions are shown. ATP increases the closing rate of Hsp90 with AMP-PNP∗ bound, and decelerates the reverse reaction, as well as the dissociation of AMP-PNP∗. Aha1 also accelerates the closing of AMP-PNP∗-bound Hsp90. In a combination of Aha1 and ATP, their effects on the closing of AMP-PNP∗-bound Hsp90 add up, whereas Aha1 prevents the decelerating effects of ATP. (b) Our observations could be caused by the depicted local rearrangements, which have been proposed to be affected by Aha1 and nucleotide binding. (Left) Reported rearrangement of the catalytic loop upon binding of Aha1 (green and dark green, superposition of the crystal structures PDB: 2CG9 (35) and 1USV (10)). (Right) Reported conformation of the nucleotide lid in the AMP-PNP or the ADP-bound crystal structures (dark and light blue, superposition of the crystal structures PDB: 2CG9 and 2WEP (36)).
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions