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Torturing Molecular Motors: Single-Molecule Studies of Viral Packaging and Flagellar Switching


Molecular motors play a central role in all known living systems. Cells use them to fight the never ending battle against entropy. Motors allow cells to organize their components in space and time to perform complex functions. In this thesis I focus on two molecular motors: the packaging motor of phi29, and the flagellar motor of Escherichia coli. These are both well-studied motors with decades of research poured into them. By leveraging past knowledge, I was able to undertake subtle studies of their function. I tortured these motors by putting them in sub-optimal operating conditions to test their limits.

For the phage motor we were primarily interested in what sort of contacts it uses to engage and exert force on its DNA substrate. We queried the nature of these contacts by making a series of modified DNA substrates, with a focus on methylphosphonate substitutions that maintain the structure of DNA while removing the negative charge. Interestingly, we found that the negative charge of the phosphates was not required for the motor to translocate DNA, but that the charge stabilizes the interaction and makes the motor less likely to slip. We also discovered that the motor interacts more strongly with one of the DNA strands, and we used these modifications to identify a set of periodic contacts.

For the flagellar motor I was interested in the role of proton motive force (PMF) in changing the rotational direction of the motor, which is controlled by a complex on its cytoplasmic side called the flagellar switch. The motor is powered by PMF, but does the PMF also affect the switch? We found that in fact the switch is affected by changes in PMF. This has implications for the switching mechanism. The switch is not only affected by the chemical binding signal, but is also affected by its associations with the motor and potentially its proximity to charges on the cell membrane.

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