The researchers knew that inside the cell lie the tiny little motors called ATPases that drive the extension and retraction of the pili. One of these ATPases is the extension motor, which sticks the bacteria's leg out. The other ATPase is the retraction motor, which pulls it back in. But what wasn't clear was how the two motors were coordinated so that pushing and pulling didn't occur at the same time. That is what Redinbo and Wolfgang set out to discover.
First, they resolved the crystal structure of the Pseudomonas PilY1 protein, which other research had shown was necessary for the creation of pili. They made large amounts of the protein, coaxed it out of solution so that it formed a crystal, and then put the crystal under intense x-ray beams through a process called x-ray diffraction that resulted in a series of spots. Based on the spots, the researchers calculated what the protein looked like. When they studied the structure, one particular site the binding site of a calcium atom looked like it could be important for the function of the protein. So the researchers began to tinker with the site, looking to see if the changes they made affected the protein's behavior.
When they changed the protein so it could no longer bind calcium, the bacteria couldn't make any legs. When they fooled the protein into thinking it was forever bound to calcium, the bacteria made legs but couldn't retract them, essentially becoming paralyzed. The results suggested that the protein has to bind calcium to make legs, but it also has to be able to let go of the calcium to pull the legs back in.
"We found it pretty remarkable that the binding of a single atom to a protein that is outside the cell is sufficient to tell these motors that are inside t
|Contact: Tom Hughes|
University of North Carolina School of Medicine