Single-Molecule Protein Unfolding and Translocation by an ATP-Fueled Proteolytic Machine

Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Cell (Impact Factor: 32.24). 04/2011; 145(2):257-67. DOI: 10.1016/j.cell.2011.03.036
Source: PubMed


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.

Full-text preview

Available from:
  • Source
    • "A ring-shaped hexamer of the ClpX ATPase first recognizes and denatures folded protein substrate, then translocates the unfolded polypeptide into the barrel-shaped ClpP peptidase for degradation. In recent studies, dual-laser optical trapping was used to dissect the molecular events resulting in protein degradation by single molecules of ClpXP (Aubin-Tam et al. 2011, Maillard et al. 2011). This technique allows for the detection of small events (nm scale) with high temporal resolution (sub-ms scale). "

    Full-text · Article · Jan 2014 · Biophysical Journal
  • Source
    • "Here molecular linkages are preferably established in-situ while still being able to sustain large forces over long timescales. Different classes of linkages have been used: Antibody-antigen linkages [13], the family of Streptavidin (STV)-biotin linkages [13]–[15], covalent disulfide linkages [14] and covalent binding proteins (HaloTag [16] or SNAP-tag [17]). Each has its own strength and drawbacks. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Many applications in biosensing, biomaterial engineering and single molecule biophysics require multiple non-covalent linkages between DNA, protein molecules, and surfaces that are specific yet strong. Here, we present a novel method to join proteins and dsDNA molecule at their ends, in an efficient, rapid and specific manner, based on the recently developed linkage between the protein StrepTactin (STN) and the peptide StrepTag II (ST). We introduce a two-step approach, in which we first construct a hybrid between DNA and a tandem of two STs peptides (tST). In a second step, this hybrid is linked to polystyrene bead surfaces and Maltose Binding Protein (MBP) using STN. Furthermore, we show the STN-tST linkage is more stable against forces applied by optical tweezers than the commonly used biotin-Streptavidin (STV) linkage. It can be used in conjunction with Neutravidin (NTV)-biotin linkages to form DNA tethers that can sustain applied forces above 65 pN for tens of minutes in a quarter of the cases. The method is general and can be applied to construct other surface-DNA and protein-DNA hybrids. The reversibility, high mechanical stability and specificity provided by this linking procedure make it highly suitable for single molecule mechanical studies, as well as biosensing and lab on chip applications.
    Full-text · Article · Jan 2013 · PLoS ONE
  • Source
    • "Very fast and highly cooperative unfolding of individual substrate domains suggests a force-dependent translocation step-size of 5–8 amino acids or 1 mm and threading is interrupted by pauses that are off the main translocation pathway. The data 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 [15] [16]. 2. Evolutionary perspective: lessons from the archaeal AAA- ATPase PAN 2.1. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The 26S proteasome is a chambered protease in which the majority of selective cellular protein degradation takes place. Throughout evolution, access of protein substrates to chambered proteases is restricted and depends on AAA-ATPases. Mechanical force generated through cycles of ATP binding and hydrolysis is used to unfold substrates, open the gated proteolytic chamber and translocate the substrate into the active proteases within the cavity. Six distinct AAA-ATPases (Rpt1-6) at the ring base of the 19S regulatory particle of the proteasome are responsible for these three functions while interacting with the 20S catalytic chamber. Although high resolution structures of the eukaryotic 26S proteasome are not yet available, exciting recent studies shed light on the assembly of the hetero-hexameric Rpt ring and its consequent spatial arrangement, on the role of Rpt C-termini in opening the 20S 'gate', and on the contribution of each individual Rpt subunit to various cellular processes. These studies are illuminated by paradigms generated through studying PAN, the simpler homo-hexameric AAA-ATPase of the archaeal proteasome. The similarities between PAN and Rpts highlight the evolutionary conserved role of AAA-ATPase in protein degradation, whereas unique properties of divergent Rpts reflect the increased complexity and tighter regulation attributed to the eukaryotic proteasome.
    Full-text · Article · Jul 2011 · Biochimica et Biophysica Acta
Show more