Mechanism of motility by dynein Molecular motor protein dynein is important for many cellular processes, such as organelle transport and cell division. Dynein achieves the role by converting the chemical energy from ATP hydrolysis into mechanical movement. However, the molecular mechanism of motility by dynein is still mysterious in many aspects, part of which are due to its large size (half MDa), relatively complex structure, such as AAA ring, stalk, and linker, and the associated biochemical challenges due to three catalytic sites for ATP hydrolysis. Unanswered important questions include (1) how many ATP molecules are consumed for each processive step?; (2) does the linker actually serve as a mechanical element for the power stroke? With a goal to broaden our understanding of dynein motility in detail, I propose to answer the two unanswered important questions.
My specific aims are as follows. (1) I will investigate the rates of ATP turnover and exchange for the three major nucleotide binding sites of dynein via a novel technique where one can specifically monitor ATP binding and ADP dissociation at AAA1/AAA3/AAA4 with a native-like dynein. (2) I will test the linker power stroke hypothesis via three complementary single molecule approaches by directly observing the linker dynamics Answers to these questions will not only contribute specifically to the dynein field with better understanding of its walking mechanism but will also broaden our general understanding of protein dynamics and allosteric regulation of protein enzymes.
The motor proteins are important for multiple cellular processes, including organelle transport and cell division by walking through their cytoskeletal tracks: actin filaments for myosin and microtubules for kinesin and dynein (Vale and Milligan 2000). Investigating the molecular mechanism of dynein motility is essential for understanding their roles in the cellular processes as well as the molecular basis of the related human diseases, such as cancer and motor neuron degeneration.
