摘要：Cytoplasmic dynein is a dimeric motor protein which processively moves along microtubule. Its motor domain (head) hydrolyzes ATP and induces conformational changes of linker, stalk, and microtubule binding domain (MTBD) to trigger stepping motion. Here we applied scattering imaging of gold nanoparticle (AuNP) to visualize load-free stepping motion of processive dynein. We observed artificially-dimerized chimeric dynein, which has the head, linker, and stalk from Dictyostelium discoideum cytoplasmic dynein and the MTBD from human axonemal dynein, whose structure has been well-studied by cryo-electron microscopy. One head of a dimer was labeled with 30 nm AuNP, and stepping motions were observed with 100 μs time resolution and sub-nanometer localization precision at physiologically-relevant 1 mM ATP. We found 8 nm forward and backward steps and 5 nm side steps, consistent with on- and off-axes pitches of binding cleft between αβ-tubulin dimers on the microtubule. Probability of the forward step was 1.8 times higher than that of the backward step, and similar to those of the side steps. One-head bound states were not clearly observed, and the steps were limited by a single rate constant. Our results indicate dynein mainly moves with biased small stepping motion in which only backward steps are slightly suppressed.
