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Title page for ETD etd-07212016-163001

Type of Document Dissertation
Author Cordova, Juan Carlos
Author's Email Address jc.cordova@vanderbilt.edu
URN etd-07212016-163001
Title Molecular Mechanisms of Protein Degradation Studied at the Single Molecule Level
Degree PhD
Department Chemical Engineering
Advisory Committee
Advisor Name Title
Matthew J. Lang Committee Chair
Jamey Young Committee Member
Martin Egli Committee Member
Peter Cummings Committee Member
  • ClpXP
  • molecular motor
  • biophysics
  • single molecule
  • protease
  • optical trapping
  • fluorescence
  • degradation
  • proteolysis
Date of Defense 2016-06-28
Availability unrestricted
Enzymatic proteins catalyze biochemical reactions central to many cellular processes. Across all kingdoms of life, members of the AAA+ (ATPases associated with diverse cellular activities plus) superfamily of enzymes convert chemical energy to mechanical work through hydrolysis of the molecule adenosine triphosphate (ATP). One of the best characterized AAA+ systems is the ClpXP protease, which degrades cellular proteins targeted for disposal. As an oligomeric stacked­ring structure comprised of the AAA+ ClpX unfoldase and the ClpP peptidase, ClpXP repeatedly pulls against a targeted protein until it ultimately mechanically unfolds the substrate. Upon unfolding, ClpX translocates the polypeptide into the ClpP peptidase for degradation. Although structural and ensemble biochemical studies have established many of the operating principles of ClpXP, a detailed mechanochemical model for protein degradation remains largely unknown.

Here we employ and engineer novel single­molecule approaches to elucidate the underlying physical mechanisms of protein unfolding and polypeptide translocation by ClpXP at the single molecule level. Using a high­resolution optical trapping assay, we directly monitor degradation of homopolymer substrates to establish the kinetics of protein unfolding which are tightly linked to substrate stability near the site of ClpX pulling. During translocation, ClpXP spools polypeptides in ~1­4nm steps, even when only two of six subunits are active in ClpX mutants, suggesting a large amount of subunit cooperativity within the ClpX ring. Using these findings, we present a mechanochemical model for protein degradation by the ClpXP protease. Furthermore, we develop single­molecule fluorescence assays to report on nucleotide binding to single ClpX subunits, as well as vital structural rearrangements in the ClpX ring. These fluorescence approaches are then combined with optical trapping to simultaneously visualize ClpX conformation, nucleotide binding, and mechanical activity. Lastly, a synthetic technique for functionalizing highly stable optical trapping handles with proteins and nucleic acids is presented.

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