Adenosine triphosphate (ATP), the universal fuel of the cell, is synthesized from adenosine diphosphate (ADP) and inorganic phosphate (P(i)) by 'ATP synthase' (F(O)F(1)-ATPase). During respiration or photosynthesis, an electrochemical potential difference of protons is set up across the respective membranes. This powers the enzyme's electrical rotary nanomotor (F(O)), which drives the chemical nanomotor (F(1)) by elastic mechanical-power transmission, producing ATP with high kinetic efficiency. Attempts to understand in detail the mechanisms of torque generation in this simple and robust system have been both aided and complicated by a wealth of sometimes conflicting data.

The 2 nanomotors of rotary ATP synthase, ionmotive FO and chemically active F1, are mechanically coupled by a central rotor and an eccentric bearing. Both motors rotate, with 3 steps in F1 and 10 -15 in FO. Simulation by statistical mechanics has revealed that an elastic power transmission is required for a high rate of coupled turnover. Here, we investigate the distribution in the FOF1 structure of compliant and stiff domains. The compliance of certain domains was restricted by engineered disulfide bridges between rotor and stator, and the torsional stiffness () of unrestricted domains was determined by analyzing their thermal rotary fluctuations. A fluorescent magnetic bead was attached to single molecules of F 1 and a fluorescent actin filament to FOF1, respectively. They served to probe first the functional rotation and, after formation of the given disulfide bridge, the stochastic rotational motion. Most parts of the enzyme, in particular the central shaft in F1, and the long eccentric bearing were rather stiff (torsional stiffness > 750 pNnm). One domain of the rotor, namely where the globular portions of subunits ␥ and of F1 contact the c-ring of FO, was more compliant ( Х 68 pNnm). This elastic buffer smoothes the cooperation of the 2 stepping motors. It is located were needed, between the 2 sites where the power strokes in FO and F1 are generated and consumed.elasticity ͉ F-ATPase ͉ nanomotor

ATP is synthesized by ATP synthase (F O F 1 -ATPase). Its rotary electromotor (F O ) translocates protons (in some organisms sodium cations) and generates torque to drive the rotary chemical generator (F 1 ). Elastic power transmission between F O and F 1 is essential for smoothing the cooperation of these stepping motors, thereby increasing their kinetic efficiency. A particularly compliant elastic domain is located on the central rotor ( c 10–15 / ϵ / γ ), right between the two sites of torque generation and consumption. The hinge on the active lever on subunit β adds further compliance. It is under contention whether or not the peripheral stalk (and the “stator” as a whole) also serves as elastic buffer. In the enzyme from Escherichia coli , the most extended component of the stalk is the homodimer b 2 , a right-handed α-helical coiled coil. By fluctuation analysis we determined the spring constant of the stator in response to twisting and bending, and compared wild-type with b -mutant enzymes. In both deformation modes, the stator was very stiff in the wild type. It was more compliant if b was elongated by 11 amino acid residues. Substitution of three consecutive residues in b by glycine, expected to destabilize its α-helical structure, further reduced the stiffness against bending deformation. In any case, the stator was at least 10-fold stiffer than the rotor, and the enzyme retained its proton-coupled activity.

BackgroundAs a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures.ResultsThe selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner.ConclusionThe MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.