Protease Power Strokes Force Proteins to Unfold
Jorge Alegre-Cebollada, Pallav Kosuri, and Julio M. Fernandez
ATP-dependent proteases degrade proteins in the cytosol of cells. Two recent articles, by Aubin-Tam et al. (2011) and Maillard et al. (2011), use single-molecule optical tweezers to show directly that these molecular machines use the energy derived from ATP hydrolysis to mechanically unfold and translocate its substrates into the proteolytic chamber.
Journal: Cell
Single-Molecule Protein Unfolding and Translocation by an ATP-Fueled Proteolytic Machine
Marie-Eve Aubin-Tam, Adrian O. Olivares, Robert T. Sauer, Tania A. Baker, and Matthew J. Lang
All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5–8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP.
Journal: Cell
ClpX(P) Generates Mechanical Force to Unfold and Translocate Its Protein Substrates
Rodrigo A. Maillard, Gheorghe Chistol, Maya Sen, Maurizio Righini, Jiongyi Tan, Christian M. Kaiser, Courtney Hodges, Andreas Martin, and Carlos Bustamante
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.
Journal: Cell
Constructing polyproteins consisting of identical tandem repeats of proteins provides an unambiguous method of investigating the mechanical properties of proteins at the single-molecule level using force spectroscopy techniques. Here we report a maleimide–thiol coupling-based facile method of constructing polyproteins for single-molecule force spectroscopy studies on the mechanical properties of proteins. This method allows for the construction of polyproteins in an efficient fashion under room temperature. The resultant thioether bonds are resistant to reduction and make it possible to carry out single-molecule force spectroscopy studies under various redox conditions. This novel method complements existing polyprotein engineering methods and can be easily applied to a wide variety of proteins.
