Engineered KIF1A Constructs for Comparative Studies

The KIF1A-WT construct [adapted from Addgene #61665 (12 (link))] consists of the R. norvegicus KIF1A residues 1 to 393, followed by a GCN4 leucine zipper for dimerization and an eGFP tag. The KIF1A-SW was modified by swapping the native loop-12 (residues 288 to 308) of the KIF1A construct with the Drosophila melanogaster KHC loop-12 sequence (GNKTHIPYRD). This D. melanogaster loop-12 sequence was used because it provides a direct comparison to previous work (11 (link), 16 (link)), and it changes the charge of the loop with less sequence divergence than using loop-12 from KIF5B. Both constructs have a C-terminal His tag and were bacterially expressed and purified by nickel gravity column chromatography, as described previously (16 (link)). The elution buffer, consisting of 20 mM phosphate buffer, 500 mM sodium chloride, 500 mM imidazole,10 μM ATP, and 5 mM DTT was supplemented with 10% glycerol before flash freezing and storing at −80 °C. Concentrations were determined using GFP absorbance at 488 nm.