The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

运动蛋白动力蛋白在沿微管运动期间经历其结构域的协调构象变化。以前的单分子研究分析了动力蛋白同二聚体的 AAA 环的运动，但没有分析沿着轨道步进的远端微管结合域 (MTBD)。在这里，我们同时以纳米精度跟踪单个动力蛋白的两个 MTBD 和一个 AAA 环，因为它使用三色成像经历了数百个步骤。我们表明 AAA 环和 MTBD 并不总是同时步进并且可以采取不同大小的步进。AAA 环和 MTBD 之间运动的这种可变性导致运动过程中动力蛋白的构象状态出乎意料地大量出现。提取关于构象转换偏差的数据，我们可以准确地模拟动力蛋白步进。