Figure 2. | Examining kinesin processivity within a general gating framework

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Examining kinesin processivity within a general gating framework

Figure 2.

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Stanford University, United States; Pennsylvania State University, United States
Figure 2.
Download figureOpen in new tabFigure 2. Kinesin with as many as 3 AA inserted in the NL maintains a 1-HB ATP-waiting state.

(A) Half-site mantADP release measurements as a function of NL insert length (mean ± SE; N = 3). Upon MT binding, both DmK-WT and DmK-3AA, pre-incubated with mantADP (mantD), lose ∼50% of their initial fluorescence. The fluorescence loss exceeds 50% for constructs containing NL inserts longer than 3 AA. Inset, A 50% loss of fluorescence corresponds to dimers binding to MTs in a 1-HB state, whereas a 100% fluorescence decrease is consistent with the release of all bound mantADP (mantD) upon MT binding. (B) MantADP exchange by the tethered head as a function of NL insert length (mean ± SE; N = 3), measured by rapidly diluting mantADP·kinesin·MT complexes into nucleotide-free buffer via stopped flow. The cartoon depicts the measured reaction. The exchange rate increased significantly for constructs with NL inserts of 4 AA or more. In the insets (A and B), white shading indicates non-fluorescent, nucleotide-free heads (Ø); yellow indicates fluorescent, mantADP-bound heads.

DOI: http://dx.doi.org/10.7554/eLife.07403.004