Elucidation Of Structural States Of Dimeric Motor Domain Of Dynein Using Cys-light Construct

Abstract

Cytoplasmic dynein is a motor protein that moves unidirectionally along a microtubule utilizing energy released by ATP hydrolysis. Introduction of probes such as fluorescent dyes that report structural changes and protein-protein interactions at particular locations will help to elucidate the molecular mechanism of force generation by dynein. For the site-directed labeling, we have replaced potentially reactive cys residues in the motor domain of Dictyostelium cytoplasmic dynein (the 380kDa fragment) with other amino acid residues without much affecting its motor activities. By using this cys-light dynein, we can insert a reactive cys residue in a specific, pre-selected location and selectively label the newly introduced cys residue with a fluorescent dye. To test the usefulness of this cys-light dynein, we introduced a reactive cys residue at the stalk head or at the stalk base of the dynein motor domain and then labeled it with Cy3 or Cy5. Introduction of the reactive cys residue and the subsequent Cy3/Cy5 labeling did not significantly affect microtubule-activated ATPase activity of cys-light dynein, suggesting the successful Cy3/Cy5 labeling of the stalk head or the stalk base. We then dimerized these Cy3 and Cy5 labeled cys-light dyneins by using a hetero-dimerizer to examine if the two stalks align closely in the dimeric dynein motor domain. The FRET measurements between the Cy3 and Cy5 labels showed that the two stalks actually stay closely in the dimer.