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. 2015 Nov 23;5:17058. doi: 10.1038/srep17058

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Copyright © 2015, Macmillan Publishers Limited

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(A) The complex formation of PPH-5 and CeHsp90 was tested in a titration approach in analytical ultracentrifugation experiments. Shown are the dc/dt plots derived from Sedview from one full experimental setup. The absorption values were used to refine the concentrations in a global fit of the data. These concentrations were 4 μM CeHsp90 alone (orange), CeHsp90 +3.5 μM PPH-5 (red), +7 μM PPH-5 (violet), +14 μM PPH-5 (green), +21 μM PPH-5 (light blue), +28 μM PPH-5 (dark blue). As a control we used 7 μM PPH-5 alone (black) (B) Replot of the concentration values for free PPH-5 (black9 and CeHsp90 containing protein complexes (red) against added PPH-5. Data analysis as global fit of both curves was performed with the program Origin as described in the materials and methods section. (C) A similar titration approach as in (A) was performed for the MC-domain of CeHsp90. Titration of PPH-5 to 3.5 μM CeHsp90-MC (alone: orange, +1.4 μM PPH-5: red, +2.8 μM PPH-5: violet, +5.6 μM PPH-5: green, +12 μM PPH-5: blue, +16 μM PPH-5: marine blue) resulted in the formation of larger protein complexes. Shown again are the Sedview derived dc/dt-plots. 7 μM PPH-5 served as a control (black) (D) Replot of the concentration values for free PPH-5 (black) and in complex with CeHsp90-MC (red). Data analysis was performed as global fit of the concentrations of the individual species from the titration experiment. The red trace from (C) (3.5 μM CeHsp90-MC +1.4 μM PPH-5) was omitted as separation between the peaks could not be sufficiently observed. (E) Determination of the form factors using the stoichiometry obtained from the global fit and the differences in absorption for the sedimenting Hsp90 complexes. Form factors were determined for each of the following proteins or protein complexes: PPH-5 (red), CeHsp90 (black), CeHsp90-MC (green), PPH-5-CeHsp90 (blue), PPH-5-CeHsp90-MC (marine blue). The values for the plot of f/f0 against the molecular weight were obtained from Sedfit analysis of the experimental data set at a pre-set f/f0 parameter.