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Figure 5.
Fig. 5. Mutational analyses of crystallographic Vps4 dimer
interfaces. (a) Crystallographic Vps4 dimer interfaces.
Interface 1 is a symmetric interface between two large ATPase
domains (residue Q216 is shown in cyan), interface 2 is a
symmetric interface between two small ATPase domains (residue
L407 is shown in blue), and interface 6 is an asymmetric
interface between the large and small ATPase domains (residues
L151 and W388 are shown in orange and green, respectively). (b)
Gel-filtration chromatograms of Vps4[ΔMIT] proteins with the
following mutations: Q216A (interface 1), L407D (interface 2),
L151D (interface 6), and W388A (interface 6). Vps4[ΔMIT]
proteins used here and elsewhere contained the E233Q mutation,
which allowed ATP binding but inhibited hydrolysis. For
reference, the elution profile of the “wild-type”
Vps4[ΔMIT],[E233Q] protein is shown in red in each panel,
elution positions for monomeric (1) and dimeric (2) proteins are
shown as dotted vertical lines, and the elution positions of
molecular weight standards are shown below the chromatograms.
Vps4 protein concentrations were 150 μM in all cases. Note that
at low micromolar concentrations, the dimeric proteins exhibited
concentration-dependent mobilities (not shown), indicating that
appreciable concentrations of monomers could accumulate under
these low-protein and nonequilibrium conditions.
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