Oliver Pänke

F-ATPase: specific observation of the rotating c subunit oligomer of EFoEF1

The rotary motion in response to ATP hydrolysis of the ring of c subunits of the membrane portion, Fo, of ATP synthase, FoF1, is still under contention. It was studied with EFoEF1 (Escherichia coli) using microvideography with a fluorescent actin filament. To overcome the limited specificity of actin attachment through a Cys‐maleimide couple which might have hampered the interpretation of previous work, we engineered a ‘strep‐tag’ sequence into the C‐terminal end of subunit c. It served (a) to purify the holoenzyme and (b) to monospecifically attach a fluorescent actin filament to subunit c. EFoEF1 was immobilized on a Ni‐NTA‐coated glass slide by the engineered His‐tag at the N‐terminus of subunit β. In the presence of MgATP we observed up to five counterclockwise rotating actin filaments per picture frame of 2000 μm2 size, in some cases yielding a proportion of 5% rotating over total filaments. The rotation was unequivocally attributable to the ring of subunit c. The new, doubly engineered construct serves as a firmer basis for ongoing studies on torque and angular elastic distortions between F1 and Fo.

Viscoelastic dynamics of actin filaments coupled to rotary F-ATPase: angular torque profile of the enzyme.

ATP synthase (F(O)F(1)) operates as two rotary motor/generators coupled by a common shaft. Both portions, F(1) and F(O), are rotary steppers. Their symmetries are mismatched (C(3) versus C(10-14)). We used the curvature of fluorescent actin filaments, attached to the rotating c-ring, as a spring balance (flexural rigidity of 8. 10(-26) Nm(2)) to gauge the angular profile of the output torque at F(O) during ATP hydrolysis by F(1) (see theoretical companion article (. Biophys. J. 81:1234-1244.)). The large average output torque (50 +/- 6 pN. nm) proved the absence of any slip. Variations of the torque were small, and the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the threefold stepping and high activation barrier of the driving motor proper, the rather constant output torque implied a soft elastic power transmission between F(1) and F(O). It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate of the two counteracting and stepping motor/generators.

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

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.

Inter-subunit rotation and elastic power transmission in F0F1-ATPase.

ATP synthase (F‐ATPase) produces ATP at the expense of ion‐motive force or vice versa. It is composed from two motor/generators, the ATPase (F1) and the ion translocator (F0), which both are rotary steppers. They are mechanically coupled by 360° rotary motion of subunits against each other. The rotor, subunits γϵc 10–14, moves against the stator, (αβ)3δab 2. The enzyme copes with symmetry mismatch (C3 versus C10–14) between its two motors, and it operates robustly in chimeric constructs or with drastically modified subunits. We scrutinized whether an elastic power transmission accounts for these properties. We used the curvature of fluorescent actin filaments, attached to the rotating c ring, as a spring balance (flexural rigidity of 8·10−26 N m2) to gauge the angular profile of the output torque at F0 during ATP hydrolysis by F1. The large average output torque (56 pN nm) proved the absence of any slip. Angular variations of the torque were small, so that the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the three‐fold stepping and high activation barrier (>40 kJ/mol) of the driving motor (F1) itself, the rather constant output torque seen by F0 implied a soft elastic power transmission between F1 and F0. It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate under load of the two counteracting and stepping motors/generators.

F1-ATPase, the C-terminal End of Subunit γ Is Not Required for ATP Hydrolysis-driven Rotation

ATP hydrolysis by the isolated F1-ATPase drives the rotation of the central shaft, subunit γ, which is located within a hexagon formed by subunits (αβ)3. The C-terminal end of γ forms an α-helix which properly fits into the “hydrophobic bearing” provided by loops of subunits α and β. This “bearing” is expected to be essential for the rotary function. We checked the importance of this contact region by successive C-terminal deletions of 3, 6, 9, 12, 15, and 18 amino acid residues (Escherichia coliF1-ATPase). The ATP hydrolysis activity of a load-freeensemble of F1 with 12 residues deleted decreased to 24% of the control. EF1 with deletions of 15 or 18 residues was inactive, probably because it failed to assemble. The average torque generated by a single molecule of EF1 when loaded by a fluorescent actin filament was, however, unaffected by deletions of up to 12 residues, as was their rotational behavior (all samples rotated during 60 ± 19% of the observation time). Activation energy analysis with the ensemble revealed a moderate decrease from 54 kJ/mol for EF1(full-length γ) to 34 kJ/mol for EF1(γ-12). These observations imply that the intactness of the C terminus of subunit γ provides structural stability and/or routing during assembly of the enzyme, but that it is not required for the rotary action under load, proper.

Kinetic modelling of the proton translocating CF0CF1-ATP synthase from spinach

The rate of both ATP synthesis and hydrolysis catalysed by the thiol‐modulated and activated ATP synthase from spinach is measured as a function of all substrates including the protons inside the thylakoid lumen. The most important findings are: (1) sigmoid kinetics with respect to Hin +, (2) hyperbolic kinetics with respect to ADP, ATP and phosphate, with K m for phosphate and ADP decreasing upon increasing Hin +, (3) binding of ADP and phosphate in random order and competitive to ATP. Simulation of the complete set of experimental data is obtained by a kinetic model featuring Boyers binding‐change mechanism.

Rotation of the c Subunit Oligomer in EF0EF1 Mutant cD61N

ATP synthases (F0F1-ATPases) mechanically couple ion flow through the membrane-intrinsic portion, F0, to ATP synthesis within the peripheral portion, F1. The coupling most probably occurs through the rotation of a central rotor (subunits c10εγ) relative to the stator (subunits ab2δ(αβ)3). The translocation of protons is conceived to involve the rotation of the ring of c subunits (the c oligomer) containing the essential acidic residue cD61 against subunits ab2. In line with this notion, the mutants cD61N and cD61G have been previously reported to lack proton translocation. However, it has been surprising that the membrane-bound mutated holoenzyme hydrolyzed ATP but without translocating protons. Using detergent-solubilized and immobilized EF0F1 and by application of the microvideographic assay for rotation, we found that the c oligomer, which carried a fluorescent actin filament, rotates in the presence of ATP in the mutant cD61N just as in the wild type enzyme. This observation excluded slippage among subunit γ, the central rotary shaft, and the c oligomer and suggested free rotation without proton pumping between the oligomer and subunit a in the membrane-bound enzyme.