Engineering Heterodimeric Kinesins through Genetic Incorporation of Noncanonical Amino Acids.

Kinesins are commonly homodimers with two identical heavy chains (protomers) and play indispensable roles in many intracellular processes. Engineered heterodimeric kinesins with two distinct protomers are important tools for dissecting coordination and regulation of naturally occurring kinesin homodimers. Here, we report a chemical-biology-based approach that generates kinesin heterodimers by combining genetic incorporation of reactive noncanonical amino acids and small-molecule-based cross-linking. We verified using yeast kinesin-8/Kip3 as a model system that our method yields kinesin heterodimers of desired properties without introducing unintended motility disruption. To demonstrate the utility of our method, we engineered a crippled Kip3 heterodimer that contains both a wild-type-like protomer and a catalytically inactive one, and our results revealed that the resulting heterodimer moves on the microtubule with a significant reduction in velocity but not processivity. Due to its versatility, we expect that our method can be broadly adopted to create novel heterodimers for other kinesins and will thus greatly expand the studies on kinesin mechanisms.

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