Table 1.
Kif5A mutations that cause HSP.
Table 2.
Effects of HSP mutations on the basal and MT-Stimulated ATPase rate.
Fig 1.
Microtubule affinity in the presence of ATP.
A) Sample western blot of a single microtubule pelleting assay. S indicates the supernatant fraction and P indicates the pellet that had been resuspended in a volume of buffer equal to that of the supernatant. B) Quantitation of the average band densitometry of the pellet fraction from three separate microtubule pelleting assays for each Kif5A construct. Error bars show one standard deviation. Asterisks indicate a significant increase of kinesin in the microtubule-bound pellet fraction (p<0.05).
Fig 2.
MT affinity in the presence of AMPPNP.
MT pelleting assays were performed as described for Fig 1, except 5 mM AMPPNP was substituted in the reaction buffer. Experiments were repeated in a range of tubulin concentrations from 0 to 20 μM and the results of the densitometry analysis of the western blots produced are plotted for each Kif5A mutant. The curves shown are the best fit to the data using the exponential decay function in Origin 8 in a user free computational method. A) Curves of fraction Kif5A bound as a function of tubulin concentration for the wild type and mutant proteins. B-H) Individual Kif5A curves (WT or mutant as indicated) with mean +/- SD for each concentration of tubulin analyzed.
Fig 3.
Molecular dynamics simulations of predicted altered kinesin structure.
Images shown are the end point of a 10 ns simulation for wild type or mutant kinesin using the 4HNA structure as a starting point. Amino acids of interest are labeled in white. Intramolecular distances between potential bonding partners are shown in purple, with units of angstroms.
Table 3.
Microtubule gliding velocity.
Fig 4.
Effect of divalent cation on S203C microtubule gliding velocity.
A) Microtubule gliding velocity of the S203C mutant in buffers with the divalent cations shown. The asterisk indicates that the velocity in Mn2+ is significantly greater than the velocity in Mg2+ (p<0.01), although still significantly lower than wild type in Mg2+ (p<0.01). Note the log scale of the Y-axis. B) Microtubule gliding velocity of the S203C mutant that was purified in the presence of Mg2+ (Mg-S203C) and Mn2+ (Mn-S203C). The single asterisk indicates that the velocity of Mg-S203C in Mn2+ is significantly greater than in Mg2+ (p<0.01), while the double asterisk indicates that the velocity of Mn-S203C in Mn2+ is significantly greater than in Mg2+ (p<0.01), and also significantly greater than the velocity of Mg-S203C in Mn2+ (p<0.05). C) Histograms of the data for S203C mutants in the conditions indicated. A similar number of microtubules (n = 90–110) were analyzed in each condition.
Fig 5.
Mn2+-induced rescue of S203C activity.
A) ATPase rate of WT, Mg-S203C, and Mn-S203C in buffers containing Mg2+ (100% Mg2+), Mn2+ (100% Mn2+), or an equal ratio of Mg2+ to Mn2+ (50% Mg2+, 50% Mn2+). The asterisk indicates that the ATPase rate in the 1:1 ratio of divalent cation is significantly greater than the rate in Mg2+ alone (p<0.01). B) Histograms with Gaussian fits to the data of Mn-S203C in buffer conditions with the relative Mn2+:Mg2+ ratios as shown.