An examination of the cytoplasmic dynein stepping mechanism at the single molecule level

Rotational motions play important roles within biological processes. These motions can drive energy production as with the F1-ATP synthase or accompany domain motions during a conformational change such as the relative rotation of the large and small ribosomal subunits during protein synthesis. Studying these motions can provide insight into the mechanics of enzyme function that cannot be obtained by measuring its localization or chemical output alone. Rotational tracking can be done in the context of single molecule studies to observe enzymatic function at the single particle level. This presents an advantage over bulk solution studies because simultaneously occurring events, such as a solution of enzymes catalyzing a reaction, are not necessarily identical. By measuring the motions of a single molecule, short-lived states and rare events that would otherwise be averaged out can be detected. Here single molecule rotational tracking is utilized to examine the stepping mechanism of the cellular transport motor, cytoplasmic dynein. Cytoplasmic dynein walks along microtubules toward the minus end and is responsible for a wide range of cellular functions including cargo transport and chromosome alignment during cell division. This work employs a position and rotational tracking method, polarized total internal reflection fluorescence (polTIRF) microscopy. This technique requires a polarized fluorescent probe that is rigidly attached to the protein domain of interest and an optical system capable of measuring the orientation of such a probe. A functionalization method was developed to water-solubilize CdSe/CdS semiconductor quantum nanorods, which have polarized fluorescence emission, and coat them with the biotin binding protein, NeutrAvidin, in order to attach them to biotinylation sites within the dynein ring. A method was also developed to quantify the number and density of functional biotin binding sites on the nanorod surface and compare it to that of commercially available streptavidin quantum dots. These nanorods were attached to cytoplasmic dynein via two inserted biotinylation sites in AAA5 and AAA6 of the ring domain and rotational motions of the dynein ring were measured in real time using a home-built optical system capable of measuring both position and orientation simultaneously. These measurements revealed small, frequent ring rotations that occurred more than twice as frequently as steps along the microtubule track. The observed ring rotations are too small to be attributed to a classic powerstroke mechanism in which large-scale tilting produces forward motion, but instead support a flexible stalk model where tension between the two dynein heads, produced by conformational changes of the linker domain, results in bending of the flexible coiled-coil stalk and hinging at the microtubule binding domain