Fig. 3.

Y313 phosphomimetic substitution affects conformation of all Hsp90 domains. Hydrogen exchange mass spectrometry shows global impact of Y313E phosphomimetic substitution on Hsp90 conformation. Purified human Hsp90α (wild-type or Hsp90-Y313E) was diluted 1:20 into D₂O buffer and incubated for 30 s at 30 °C in the absence of nucleotide. The reaction was quenched with ice cold quench buffer, the protein digested with immobilized pepsin, desalted and analyzed by liquid chromatography mass spectrometry (LC–MS). Deuteron incorporation into Hsp90-Y313E minus deuteron incorporation into wild-type Hsp90 is plotted for all peptic peptides (shown is the difference between each pair of means ± standard error for n = 3 independent experiments). For each peptide, bars depicting increased exchange for Hsp90-Y313E (compared to wild-type Hsp90) extend to the right, while bars showing decreased exchange for Hsp90-Y313E relative to wild-type Hsp90 extend to the left. Red lines indicate domain boundaries. CL, charged linker. The source data file contains the mean values ± SEM of deuteron incorporation for each corresponding peptide obtained from wild-type Hsp90 and Hsp90-Y313E, as well as the difference between the two sets of means ± SEM. The values underlying the means are no longer available