
AAA(+) unfoldases denature and translocate polypeptides into associated peptidases. We report direct observations of mechanical, force-induced protein unfolding by the ClpX unfoldase from E. coli, alone, and in complex with the ClpP peptidase. ClpX hydrolyzes ATP to generate mechanical force and translocate polypeptides through its central pore. Threading is interrupted by pauses that are found to be off the main translocation pathway. ClpX's translocation velocity is force dependent, reaching a maximum of 80 aa/s near-zero force and vanishing at around 20 pN. ClpX takes 1, 2, or 3 nm steps, suggesting a fundamental step-size of 1 nm and a certain degree of intersubunit coordination. When ClpX encounters a folded protein, it either overcomes this mechanical barrier or slips on the polypeptide before making another unfolding attempt. Binding of ClpP decreases the slip probability and enhances the unfolding efficiency of ClpX. Under the action of ClpXP, GFP unravels cooperatively via a transient intermediate.
Adenosine Triphosphatases, Protein Denaturation, Protein Folding, Protein Transport, Biochemistry, Genetics and Molecular Biology(all), Escherichia coli Proteins, Green Fluorescent Proteins, Escherichia coli, ATPases Associated with Diverse Cellular Activities, Endopeptidase Clp, Biomechanical Phenomena, Molecular Chaperones
Adenosine Triphosphatases, Protein Denaturation, Protein Folding, Protein Transport, Biochemistry, Genetics and Molecular Biology(all), Escherichia coli Proteins, Green Fluorescent Proteins, Escherichia coli, ATPases Associated with Diverse Cellular Activities, Endopeptidase Clp, Biomechanical Phenomena, Molecular Chaperones
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