SUMMARY 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.

Summary ATP-dependent proteases are vital to maintain cellular protein homeostasis, but the mechanisms by which these machines couple ATP hydrolysis to mechanical protein unfolding and translocation remain unclear. Here, we study the mechanisms of force generation and inter-subunit coordination in the ClpXP protease from E. coli. We identify phosphate release as the force-generating step of the ATPase cycle and reveal that ClpXP translocates substrate polypeptides by highly coordinated conformational changes in up to four ATPase subunits. To unfold stable substrates like GFP, ClpXP must use this maximum successive firing capacity. The dwell duration between individual bursts of translocation is constant and governed by an internal clock, regardless of the number of translocating subunits, implying that ClpXP operates with constant “rpm” but different “gears”.

molecular analysis with sensitivity and selectivity. Here we describe an innovative biophysical solution to prevent hemolysis in blood plasma separation devices for precision molecular diagnostics. In order to avoid biochemical interferences, RBCs are exposed to both fluidic drag force and gravitational force after the computation of transition fluid velocity from vertical up-flow to sedimentation was in the range of 1.3 to 2.5 mm/sec in the design of microfluidic separation device. A membrane filter for filtration was positioned on top of a vertical up-flow channel to reduce clogging of RBCs by gravity-assisted cells sedimentation. As a result, separated plasma volume was increased about 4-fold (2.4 mL plasma after 20 min with 38 % hematocrit human whole blood) and hemoglobin concentration in separated plasma was decreased about 90 % due to the prevention of RBCs hemolysis in comparison to a filter-in-bottom configuration. On-chip plasma contains~90 % of protein and~100 % of nucleic acids compared to off-chip centrifuged plasma, showing comparable target molecules recovery. This simple and reliable blood plasma separation platform can be easily integrated with downstream detection module for sample-toanswer POC diagnostics.