FIGURE 5.

Pre-steady-state analysis of NSF ATP hydrolysis and SNARE disassembly. A, NSF hydrolyzes ATP in the NSF·αSNAP·SC (20 S) complex, leading to SC disassembly (i), and outside the 20 S complex (ii and iii). B, SC disassembly in the presence (filled circles) and absence (open circles) of Mg²⁺ (n = 3; error bars show S.E.). C, ATP hydrolysis in the presence (filled circles) and absence (open circles) of αSNAP₃. C and D, a representative pre-steady-state experiment ([NSF] = 1.96 μm, [αSNAP₃] = 100 nm, [SC] = 750 nm) (n = 3; error bars show S.E.) used to obtain reaction rates. D, pre-steady-state disassembly and ATP hydrolysis experiments were performed at different SC concentrations (38–900 nm), and the rate constant for ATP hydrolysis versus SC concentration is plotted with the full data from Table 4. The percentage of αSNAP₃ in the αSNAP₃·SC complex in each experiment (dashed line, right y axis) was calculated from experimental protein concentrations and the independently determined dissociation constant of K_d^αSNAP·SC = 100 nm (see “Experimental Procedures”) (error bars show propagated uncertainty in the linear regression of ATP hydrolysis and SC disassembly measurements in B and C above). E, the number of ATPs hydrolyzed per SC disassembly are plotted versus SC concentration of each experiment (data points, left y axis). The percentage of αSNAP₃·SC fits the data points well (dashed line, right y axis). Error bars, propagated uncertainty in the calculation of ATP per SC.