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.

中文翻译：

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通过非规范氨基酸的遗传掺入工程异源二聚体驱动蛋白。

驱动蛋白通常是具有两个相同的重链（启动子）的同型二聚体，并且在许多细胞内过程中起着不可或缺的作用。具有两个不同的启动子的工程异二聚体驱动蛋白是剖析天然存在的驱动蛋白同型二聚体的协调和调控的重要工具。在这里，我们报告了一种基于化学生物学的方法，该方法通过将反应性非规范氨基酸的遗传掺入和基于小分子的交联相结合来生成驱动蛋白异二聚体。我们验证了使用酵母驱动蛋白8 / Kip3作为模型系统，我们的方法可产生具有所需特性的驱动蛋白异二聚体，而不会引起意想不到的运动破坏。为了证明我们方法的实用性，我们设计了一个残缺的Kip3异二聚体，该异源二聚体既包含野生型启动子，也具有催化惰性的启动子，我们的结果表明，生成的异二聚体在微管上移动，但速度显着降低，但合成能力却没有降低。由于它的多功能性，我们希望我们的方法可以被广泛采用来创建其他驱动蛋白的新型异二聚体，从而极大地扩展对驱动蛋白机理的研究。