Effect of nucleotides on the conformation of Ssc1 from Single-molecule

molecule FRET measurements

As the ensemble measurements indicate an average of FRET-efficiencies of all the molecules being measured, changes in distance as read out from the PR is only indicative of mean changes in distance. Single molecule FRET measurements are ideal in obtaining insights into the conformational distribution and heterogeneity. Towards elucidation of the conformational distribution, single particle FRET (SpFRET) measurements using PIE (Pulsed Interleaved Excitation) were performed (Muller et al., 2005).

For the distance vector 341-448, an unambiguous uni-modal distribution was observed upon ATP binding, suggesting majority of the molecules in the domain-docked state. Peak values of a Gaussian fit to the FRET efficiency distribution was 0.90 suggesting very compact state of majority of molecules. On the contrary, the ADP bound state was very broad and after Gaussian fitting two peaks were obtained one at 0.40 and other at 0.84 (Figure 3.45). This indicates that in the ADP-bound state the domains are flexible, populating conformations containing domain-undocked molecules as well as domain- docked molecules.

Figure 3.44: Changes in distance between the lid domain and base of PBD of Ssc1 and DnaK as probed by intra-molecular FRET experiments. Fluorescence spectrum of 30nM of double labeled Ssc1 (448,590) and DnaK (458,563) were obtained after exciting the donor fluorophore at 530nm in presence of 2mM ATP and 2mM ADP. Donor fluorescence was normalized to 1 to compare the PR in presence of different nucleotides.

91

In case of DnaK (318,425), for the distance vector 318-425, a bi-modal distribution was observed upon ATP binding. Peak values of a Gaussian fit to the FRET efficiency distribution were at 0.15 and at 0.80 which suggested a very compact state of majority of the molecules along with a small subpopulation of domain- undocked molecules. This distribution indicated some flexibility of the domains in the ATP state. On the contrary, the ADP bound state was clearly unimodal in distribution and after Gaussian fitting, a peak was obtained at 0.15 (Figure 3.46). Such a distribution is different from what was obtained for Ssc1 and suggests that the domains are fully undocked and are separated by a large distance in case of DnaK.

Figure 3.45: Nucleotide induced changes in inter-domain distance of Ssc1 as probed by SpFRET measurements. To obtain Single-molecule FRET measurements of double labeled Ssc1 (341,448) the protein was diluted in the appropriate buffer (as mentioned in the materials and methods) to a final concentration of 60 pM, containing either 2mM ATP or 2mM ADP. Representative histograms of two different nucleotide conditions are shown. Peak values of a Gaussian fit to the FRET efficiency distributions (fE) are indicated.

92

For the distance vector 448-590 in Ssc1 (448,590) probing for the movements of the α- helical lid in respect to the base of the PBD, a unimodal distribution was observed upon ATP binding. Peak values of a Gaussian fit to the FRET efficiency distribution was 0.15 suggesting lid-open state of majority of molecules. On the contrary, the distribution for ADP bound state was very broad and it was not possible to the fit the fE distribution to Gaussian fits (Figure 3.47). The flexibility of the lid in the ADP-bound state hinted that the lid was not firmly locked on to PBD, as envisioned for canonical Hsp70s.

Figure 3.46: Nucleotide induced changes in inter-domain distance of DnaK as probed by SpFRET measurements. To obtain Single-molecule FRET measurements of double labeled DnaK (318,425) the protein was diluted in the appropriate buffer (as mentioned in the materials and methods) to a final concentration of 60 pM, containing either 2mM ATP or 2mM ADP. Representative histograms of two different nucleotide conditions are shown. Peak values of a Gaussian fit to the FRET efficiency distributions (fE) are indicated.

Figure 3.47: Nucleotide induced changes in distance between lid domain and base of the PBD of Ssc1 as probed by SpFRET measurements. To obtain Single-molecule FRET measurements of double labeled Ssc1 (448,590) the protein was diluted in the appropriate buffer (as mentioned in the materials and methods) to a final concentration of 60 pM, containing either 2mM ATP or 2mM ADP. Representative histograms of two different nucleotide conditions are shown. Peak values of a Gaussian fit to the FRET efficiency distributions (fE) are indicated.

93

Similar observations were made for the 425-563 in DnaK (425,563). A uni-modal distribution was observed upon ATP binding. Peak values of a Gaussian fit to the FRET efficiency distribution was 0.20. Like Ssc1, the distribution of fE for the ADP bound state was very broad and could be fit to bimodal Gaussian distribution with one peak at 0.16 and another at 0.58 suggesting large flexibility of the lid in the ADP-bound state(Figure 3.48).

Thus, with the ensemble and SpFRET analyses, the nucleotide dependent changes in conformations of Ssc1 and its bacterial homologue DnaK could be probed. Using the same assay developed, we went further to look for effects of J domain co-chaperone and substrate on the conformations of Ssc1 in respect to its functional chaperone cycle. Simultaneously, the same experiments were performed with DnaK to compare similarities in two systems.

Figure 3.48: Nucleotide induced changes in distance between lid domain and base of the PBD of DnaK as probed by SpFRET measurements. To obtain Single-molecule FRET measurements of double labeled DnaK (458,563) the protein was diluted in the appropriate buffer (as mentioned in the materials and methods) to a final concentration of 60 pM, containing either 2mM ATP or 2mM ADP. Representative histograms of two different nucleotide conditions are shown. Peak values of a Gaussian fit to the FRET efficiency distributions (fE) are indicated.

94