Figure 9.

Summary of methodology and major findings. Hsp104 monomer and hexamer with ADP or ATPγS were subjected to increasing exposure times of synchrotron-generated X-rays. Mass spectrometer analysis of the samples yielded oxidative modification rates for pepsin-derived peptides, and MS² data identified specific sites of modification. Using the modification rates as a proxy for solvation, Hsp104 variants were designed with substitutions in residues found to be highly solvated or in regions that displayed large changes in solvation across the different states. Activity of these Hsp104 variants was then assessed using in vitro and in vivo assays to determine their role in Hsp104 function.