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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: Nat Struct Mol Biol. 2015 May 4;22(6):492–498. doi: 10.1038/nsmb.3015

Figure 2. Vps4 completely unfolds ESCRT-III substrate in the course of disassembly.

Figure 2

(a) Folded (F) to unfolded (U) conversion for the peptides in the presence of Vps4, color coded as in the Vps24 structural model shown in the inset. (b-d) Mass spectra of selected peptides from helices α1 (b), α2 (c), and α4 (d) of Vps24-2 monomers (left), filaments (middle), or filament plus Vps4 (right). Controls and time points are indicated. Arrows above the spectra indicate regions of the spectrum representing EX2 and EX1 behavior. (e-g) Peak-width analysis of the selected peptides at 5, 10, 20, 30, 40, 60 s and full deuteration (TD) for selected peptides from helices α1 (e), α2 (f), and α4 (g). Open circles, Vps24-2 monomers; filled squares, Vps24-2 filaments; triangles, Vps24-2 filaments plus Vps4. The grey bar denotes the 2 Da peak-width band for peptides undergoing EX2 kinetics 41. (h) SDS-PAGE gel and quantification bar chart of Vps4WT-mediated disassembly of Vps24-2 filaments as analyzed by sedimentation. Pellet (P) and supernatant (S) fractions were analyzed by SDS-PAGE. Error bars are the s. d. of three experiments. WT, wild-type.