Mechanistic studies of a AAA+ protease
Author(s)Nager, Andrew R. (Andrew Ross)
Mechanistic studies of a AAA plus protease
Massachusetts Institute of Technology. Department of Biology.
Robert T. Sauer.
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AAA+ proteases are present in all branches of life and responsible for the energy-dependent degradation of most cytosolic proteins. Substrates for AAA+ proteases are unfolded and translocated into a compartmental peptidase. The requirement for protein unfolding raises several questions. How easily are proteins unfolded within the native environment of a cell? Are some proteins more difficult to unfold than others, and, if so, why? How do AAA+ ATPases convert the chemical energy of ATP binding and hydrolysis into mechanical unfolding and translocation? ClpXP is a AAA+ protease that consists of the hexameric ClpX unfoldase and polypeptide translocase and the ClpP compartmental peptidase. ClpX binds a substrate by an unstructured degradation tag and then, by multiple rounds of ATP-binding and hydrolysis, unfolds and translocates the substrate into the proteolytic chamber of ClpP. To study the features that allow a protein to resist unfolding, I investigate the degradation of degron-tagged Green Fluorescent Protein (GFP; Chapter 2). By engineering GFP substrates, I determine the steps of GFP unfolding and how structure local to the degron can hinder ClpX-mediated unfolding. In later chapters, my collaborators and I use ensemble and single-molecule fluorescent assays to study the mechanochemical cycle of ClpX6 . By these assays, we observe that subunits adopt unique classes which differ in structure and nucleotide binding and hydrolysis, subunit classes switch in a thermally-driven probabilistic fashion that is decoupled from the chemical cycle, and ClpX 6 form a staircase architecture similar to AAA+ helicases.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, February 2013.Cataloged from PDF version of thesis. "December 2012."Includes bibliographical references.
DepartmentMassachusetts Institute of Technology. Department of Biology.
Massachusetts Institute of Technology