Elastic deformations of the rotary double motor of single FoF1-ATP synthases detected in real time by Förster resonance energy transfer

Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, Förster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8 nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. F_OF₁-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary F_O motor in the Escherichia coli enzyme is connected to a 3-stepped F₁ motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual F_OF₁-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

A chemical thermodynamic model of motor enzymes unifies chemical-Fx and powerstroke models

Molecular motors play a central role in many biological processes, ranging from pumping blood and breathing to growth and wound healing. Through motor-catalyzed chemical reactions, these nanomachines convert the chemical free energy from ATP hydrolysis into two different forms of mechanical work. Motor enzymes perform reversible work, w_rev, through an intermediate step in their catalyzed reaction cycle referred to as a working step, and they perform Fx work when they move a distance, x, against a force, F. In a powerstroke model, w_rev is performed when the working step stretches a spring within a given motor enzyme. In a chemical-Fx model, w_rev is performed in generating a conserved Fx potential defined external to the motor enzyme. It is difficult to find any common ground between these models even though both have been shown to account for mechanochemical measurements of motor enzymes with reasonable accuracy. Here, I show that, by changing one simple assumption in each model, the powerstroke and chemical-Fx model can be reconciled through a chemical thermodynamic model. The formal and experimental justifications for changing these assumptions are presented. The result is a unifying model for mechanochemical coupling in motor enzymes first presented by A.V. Hill in 1938 that is consistent with single-molecule structural and mechanical data.     

Reversible control of F1-ATPase rotational motion using a photochromic ATP analog at the single molecule level

Motor enzymes such as F₁-ATPase and kinesin utilize energy from ATP for their motion. Molecular motions of these enzymes are critical to their catalytic mechanisms and were analyzed thoroughly using a single molecule observation technique. As a tool to analyze and control the ATP-driven motor enzyme motion, we recently synthesized a photoresponsive ATP analog with a p-tert-butylazobenzene tethered to the 2′ position of the ribose ring. Using cis/trans isomerization of the azobenzene moiety, we achieved a successful reversible photochromic control over a kinesin-microtubule system in an in vitro motility assay. Here we succeeded to control the hydrolytic activity and rotation of the rotary motor enzyme, F₁-ATPase, using this photosensitive ATP analog. Subsequent single molecule observations indicated a unique pause occurring at the ATP binding angle position in the presence of cis form of the analog.     

The coupled chemomechanics of the F1-ATPase molecular motor

The enzyme F₁-ATPase is a rotary nanomotor in which the central γ subunit rotates inside the cavity made of α₃β₃ subunits. The experiments showed that the rotation proceeds in steps of 120° and each 120° step consists of 80° and 40° substeps. Here the Author proposes a stochastic wave mechanics of the F₁-ATPase motor and combines it with the structure-based kinetics of the F₁-ATPase to form a chemomechanic coupled model. The model can reproduce quantitatively and explain the experimental observations about the F₁ motor. Using the model, several rate-limited situations about γ subunit rotation are proposed, the effects of the friction and the load on the substeps are investigated and the chemomechanic coupled time during ATP hydrolysis cycle is determined.     

Histochemical study of an unusual cat intrafusal muscle fiber

Summary A cat tenuissimus muscle spindle that contained two long chain intrafusal fibers in its distal pole is described. One of the fibers (1 c₁) had a histochemical profile (ATPase, NADH-TR, ChE reactions) of the kind which is characteristic for long chain fibers. The other fiber (1 c₂) consisted of two separate segments. The inner 1 c₂ segment included the sensory equatorial region and was histochemically normal. The outer 1 c₂ segment carried a motor plate, and did not stain for NADH-TR in the same way as the inner 1 c₂ segment and the 1 c₁ fiber. It is suggested that the unusual enzyme staining properties of the outer 1 c₂ segment stemmed from its lack of sensory innervation, a situation which may have permitted the full expression of influences mediated by its motor nerve supply.     

A Mechanism of Modulating the Direction of Flagellar Rotation in Bacteria by Fumarate and Fumarate Reductase

Fumarate, an electron acceptor in anaerobic respiration of Escherichia coli, has an additional function of assisting the flagellar motor to shift from counterclockwise to clockwise rotation, with a consequent modulation of the bacterial swimming behavior. Fumarate transmits its effect to the motor via the fumarate reductase complex (FrdABCD), shown to bind to FliG—one of the motor’s switch proteins. How binding of the FrdABCD respiratory enzyme to FliG enhances clockwise rotation and how fumarate is involved in this activity have remained puzzling. Here we show that the FrdA subunit in the presence of fumarate is sufficient for binding to FliG and for clockwise enhancement. We further demonstrate by in vitro binding assays and super-resolution microscopy in vivo that the mechanism by which fumarate-occupied FrdA enhances clockwise rotation involves its preferential binding to the clockwise state of FliG (FliG_cw). Continuum electrostatics combined with docking analysis and conformational sampling endorsed the experimental conclusions and suggested that the FrdA–FliG_cw interaction is driven by the positive electrostatic potential generated by FrdA and the negatively charged areas of FliG. They further demonstrated that fumarate changes FrdA’s conformation to one that can bind to FliG_cw. These findings also show that the reason for the failure of the succinate dehydrogenase flavoprotein SdhA (an almost-identical analog of FrdA shown to bind to FliG equally well) to enhance clockwise rotation is that it has no binding preference for FliG_cw. We suggest that this mechanism is physiologically important as it can modulate the magnitude of ΔG⁰ between the clockwise and counterclockwise states of the motor to tune the motor to the growth conditions of the bacteria.     

Na,K-ATPase as A Brownian Motor: Electric Field-InducedConformational Fluctuation Leads to Uphill Pumping of Cation inthe Absence of ATP

Na,K-ATPase uses chemical bond energy of ATP to pump K⁺ into, andNa⁺ out of a cell. Both are uphill transports. During the catalyticcycle the enzyme alternates between two conformational states, E₁ andE₂. This communication describes an experiment, which employs electricfield to drive oscillation or fluctuation of enzyme conformation betweenthe E₁ and the E₂ states. It is shown that the field-inducedconformational oscillation or fluctuation leads to uphill pumping of thecation by the enzyme without consumption of ATP. Biochemical specificityof the catalysis is preserved. Data indicate that Na,K-ATPase can harvestenergy from the applied electric field to perform chemical work, and aratchet mechanism is inherent in this energy transduction process. ATheory of Electroconformational Coupling (TEC) that embodies essentialfeatures of the Brownian Ratchet successfully simulates the field-frequencyand field-amplitude optima and other features of the ion pumping activity.A four-state TEC motor can achieve high efficiency of the energytransduction, asymptotically reaching 100% under the optimal condition.Pumping by ion rectification fails to reach high efficiency. The TECconcept is also mused to understand other biological motors and engines.     

Coupling Proton Movement to ATP Synthesis in the Chloroplast ATP Synthase

The chloroplast F₀F₁-ATP synthase-ATPase is a tiny rotary motor responsible for coupling ATP synthesis and hydrolysis to the light-driven electrochemical proton gradient. Reversible oxidation/reduction of a dithiol, located within a special regulatory domain of the γ subunit of the chloroplast F₁ enzyme, switches the enzyme between an inactive and an active state. This regulatory mechanism is unique to the ATP synthases of higher plants and its physiological significance lies in preventing nonproductive depletion of essential ATP pools in the dark. The three-dimensional structure of the chloroplast F₁ gamma subunit has not yet been solved. To examine the mechanism of dithiol regulation, a model of the chloroplast gamma subunit was obtained through segmental homology modeling based on the known structures of the mitochondrial and bacterial γ subunits, together with de novo construction of the unknown regulatory domain. The model has provided considerable insight into how the dithiol might modulate catalytic function. This has, in turn, suggested a mechanism by which rotation of subunits in F₀, the transmembrane proton channel portion of the enzyme, can be coupled, via the ε subunit, to rotation of the γ subunit of F₁ to achieve the 120° (or 90°+30°) stepping action that is characteristic of F₁ γ subunit rotation.     

Rotation of the γ Subunit in F1-ATPase; Evidence That ATP Synthase Is a Rotary Motor Enzyme

ATP-dependent, azide-sensitive rotation of the subunit relative to the ₃₃ hexagonal ring of ATP synthase was observed with a single molecule imaging system. Thus, ATP synthase is a rotary motor enzyme, the first ever found.     

F-ATP-ase of Escherichia coli membranes: The ubiquitous MgADP-inhibited state and the inhibited state induced by the ε–subunit's C-terminal domain are mutually exclusive

ATP synthases are important energy-coupling, rotary motor enzymes in all kingdoms of life. In all F-type ATP synthases, the central rotor of the catalytic F₁ complex is composed of the γ subunit and the N-terminal domain (NTD) of the ε subunit. In the enzymes of diverse bacteria, the C-terminal domain of ε (εCTD) can undergo a dramatic conformational change to trap the enzyme in a transiently inactive state. This inhibitory mechanism is absent in the mitochondrial enzyme, so the εCTD could provide a means to selectively target ATP synthases of pathogenic bacteria for antibiotic development. For Escherichia coli and other bacterial model systems, it has been difficult to dissect the relationship between ε inhibition and a MgADP-inhibited state that is ubiquitous for F_OF₁ from bacteria and eukaryotes. A prior study with the isolated catalytic complex from E. coli, EcF₁, showed that these two modes of inhibition are mutually exclusive, but it has long been known that interactions of F₁ with the membrane-embedded F_O complex modulate inhibition by the εCTD. Here, we study membranes containing EcF_OF₁ with wild-type ε, ε lacking the full εCTD, or ε with a small deletion at the C-terminus. By using compounds with distinct activating effects on F-ATP-ase activity, we confirm that εCTD inhibition and ubiquitous MgADP inhibition are mutually exclusive for membrane-bound E. coli F-ATP-ase. We determine that most of the enzyme complexes in wild-type membranes are in the ε-inhibited state (>50%) or in the MgADP-inhibited state (30%).     

Velocity of translation of single actin filaments (AF) by myosin heads from antigen-sensitized airway smooth muscle

We have previously reported increased velocity of shortening (V_o) in the sensitized airway (0.36 1_o/s, ± SE) smooth muscle compared to the control (0.26 1_o/s, ± 0.017 SE) and subsequent experiments indicated this was due to increased phosphorylation of the 20 kDa myosin light chain resulting from increased total myosin light chain kinase activity. The motility assay technique described by Kron and Spudich was employed to determine whether additionally the molecular motor (actomyosin crossbridge) itself was altered in airway smooth muscle by ragweed pollen sensitization. The motility assay measures the velocity of actin filament translation by myosin molecules. The negative results of the motility assay were valuable in determining that the pathogenesis of allergic bronchospasm is not at contractile protein level but at regulatory enzyme level.     

Functional halt positions of rotary FOF1-ATPase correlated with crystal structures

The F_OF₁-ATPase is a rotary molecular motor. Driven by ATP-hydrolysis, its central shaft rotates in 80° and 40° steps, interrupted by catalytic and ATP-waiting dwells. We recorded rotations and halts by means of microvideography in laboratory coordinates. A correlation with molecular coordinates was established by using an engineered pair of cysteines that, under oxidizing conditions, formed zero-length cross-links between the rotor and the stator in an orientation as found in crystals. The fixed orientation coincided with that of the catalytic dwell, whereas the ATP waiting dwell was displaced from it by +40°. In crystals, the convex side of the cranked central shaft faces an empty nucleotide binding site, as if holding it open for arriving ATP. Functional studies suggest that three sites are occupied during a catalytic dwell. Our data imply that the convex side faces a nucleotide-occupied rather than an empty site. The enzyme conformation in crystals seems to differ from the conformation during either dwell of the active enzyme. A revision of current schemes of the mechanism is proposed.     

Present State and Future Perspectives of Prostaglandins as a Differentiation Factor in Motor Neurons

Spinal motor neurons have the longest axons that innervate the skeletal muscles of the central nervous system. Motor neuron diseases caused by spinal motor neuron cell death are incurable due to the unique and irreplaceable nature of their neural circuits. Understanding the mechanisms of neurogenesis, neuritogenesis, and synaptogenesis in motor neurons will allow investigators to develop new in vitro models and regenerative therapies for motor neuron diseases. In particular, small molecules can directly reprogram and convert into neural stem cells and neurons, and promote neuron-like cell differentiation. Prostaglandins are known to have a role in the differentiation and tissue regeneration of several cell types and organs. However, the involvement of prostaglandins in the differentiation of motor neurons from neural stem cells is poorly understood. The general cell line used in research on motor neuron diseases is the mouse neuroblastoma and spinal motor neuron fusion cell line NSC-34. Recently, our laboratory reported that prostaglandin E₂ and prostaglandin D₂ enhanced the conversion of NSC-34 cells into motor neuron-like cells with neurite outgrowth. Moreover, we found that prostaglandin E₂-differentiated NSC-34 cells had physiological and electrophysiological properties of mature motor neurons. In this review article, we provide contemporary evidence on the effects of prostaglandins, particularly prostaglandin E₂ and prostaglandin D₂, on differentiation and neural conversion. We also discuss the potential of prostaglandins as candidates for the development of new therapeutic drugs for motor neuron diseases.

     

Regulation of the F1F0-ATP Synthase Rotary Nanomotor in its Monomeric-Bacterial and Dimeric-Mitochondrial Forms

The F₁F₀-adenosine triphosphate (ATP) synthase rotational motor synthesizes most of the ATP required for living from adenosine diphosphate, Pi, and a proton electrochemical gradient across energy-transducing membranes of bacteria, chloroplasts, and mitochondria. However, as a reversible nanomotor, it also hydrolyzes ATP during de-energized conditions in all energy-transducing systems. Thus, different subunits and mechanisms have emerged in nature to control the intrinsic rotation of the enzyme to favor the ATP synthase activity over its opposite and commonly wasteful ATPase turnover. Recent advances in the structural analysis of the bacterial and mitochondrial ATP synthases are summarized to review the distribution and mechanism of the subunits that are part of the central rotor and regulate its gyration. In eubacteria, the ε subunit works as a ratchet to favor the rotation of the central stalk in the ATP synthase direction by extending and contracting two α-helixes of its C-terminal side and also by binding ATP with low affinity in thermophilic bacteria. On the other hand, in bovine heart mitochondria, the so-called inhibitor protein (IF₁) interferes with the intrinsic rotational mechanism of the central γ subunit and with the opening and closing of the catalytic β-subunits to inhibit its ATPase activity. Besides its inhibitory role, the IF₁ protein also promotes the dimerization of the bovine and rat mitochondrial enzymes, albeit it is not essential for dimerization of the yeast F₁F₀ mitochondrial complex. High-resolution electron microscopy of the dimeric enzyme in its bovine and yeast forms shows a conical shape that is compatible with the role of the ATP synthase dimer in the formation of tubular the cristae membrane of mitochondria after further oligomerization. Dimerization of the mitochondrial ATP synthase diminishes the rotational drag of the central rotor that would decrease the coupling efficiency between rotation of the central stalk and ATP synthesis taking place at the F₁ portion. In addition, F₁F₀ dimerization and its further oligomerization also increase the stability of the enzyme to natural or experimentally induced destabilizing conditions.     

A Globular, Not Asymmetric, Form of Acetylcholinesterase Is Expressed in Chick Motor Neurons: Down-Regulation Toward Maturity and After Denervation

Abstract: In vertebrate neuromuscular junctions, the postsynaptic specializations include the accumulation of acetylcholinesterase (AChE) at the synaptic basal lamina and the muscle fiber. Several lines of evidence indicate that the presynaptic motor neuron is able to synthesize and secrete AChE at the neuromuscular junctions. By using anti-AChE catalytic subunit, anti-butyrylcholinesterase (BuChE) catalytic subunit, and anti-AChE collagenous tail monoclonal antibodies, we demonstrated that the motor neurons of chick spinal cord expressed AChE in vivo and the predominant AChE was the globular form of the enzyme. Neither asymmetric AChE nor BuChE was detected in the motor neurons. The molecular mass of AChE catalytic subunit in the motor neuron was ∼105 kDa, which was similar to that of the globular enzyme from low-salt extracts of muscle; both of them were ∼5 kDa smaller than the asymmetric AChE from high-salt extracts of muscle. The level of AChE expression in the motor neurons decreased, as found by immunochemical and enzymatic analysis, during the different stages of the chick's development and after nerve lesion. Thus, the AChE activity at the neuromuscular junctions that is contributed by the presynaptic motor neurons is primarily the globular, not the asymmetric, form of the enzyme, and these contributions decreased toward maturity and after denervation.     

The role of a cytoplasmic loop of MotA in load‐dependent assembly and disassembly dynamics of the MotA/B stator complex in the bacterial flagellar motor

The proton‐driven flagellar motor of Salmonella enterica can accommodate a dozen MotA/B stators in a load‐dependent manner. The C‐terminal periplasmic domain of MotB acts as a structural switch to regulate the number of active stators in the motor in response to load change. The cytoplasmic loop termed MotA_C is responsible for the interaction with a rotor protein, FliG. Here, to test if MotA_C is responsible for stator assembly around the rotor in a load‐dependent manner, we analyzed the effect of MotA_C mutations, M76V, L78W, Y83C, Y83H, I126F, R131L, A145E and E155K, on motor performance over a wide range of external load. All these MotA_C mutations reduced the maximum speed of the motor near zero load, suggesting that they reduce the rate of conformational dynamics of MotA_C coupled with proton translocation through the MotA/B proton channel. Dissociation of the stators from the rotor by decrease in the load was facilitated by the M76V, Y83H and A145E mutations compared to the wild‐type motor. The E155K mutation reduced the number of active stators in the motor from 10 to 6 under extremely high load. We propose that MotA_C is responsible for load‐dependent assembly and disassembly dynamics of the MotA/B stator units.     

Combining Diffusion Tensor Imaging and Gray Matter Volumetry to Investigate Motor Functioning in Chronic Stroke

Motor impairment after stroke is related to the integrity of the corticospinal tract (CST). However, considerable variability in motor impairment remains unexplained. To increase the accuracy in evaluating long-term motor function after ischemic stroke, we tested the hypothesis that combining diffusion tensor imaging (DTI) and gray matter (GM) volumetry can better characterize long-term motor deficit than either method alone in patients with chronic stroke. We recruited 31 patients whose Medical Research Council strength grade was ≤ 3/5 in the extensor muscles of the affected upper extremity in the acute phase. We used the Upper Extremity Fugl-Meyer (UE-FM) assessment to evaluate motor impairment, and as the primary outcome variable. We computed the fractional anisotropy ratio of the entire CST (CST_ratio) and the volume of interest ratio (VOI_ratio), between ipsilesional and contralesional hemispheres, to explain long-term motor impairment. The results showed that CST_ratio, VOI_ratio of motor-related brain regions, and VOI_ratio in the temporal lobe were correlated with UE-FM. A multiple regression model including CST_ratio and VOI_ratio of the caudate nucleus explained 40.7% of the variability in UE-FM. The adjusted R2 of the regression model with CST_ratio as an independent variable was 29.4%, and that of using VOI_ratio of the caudate nucleus as an independent variable was 23.1%. These results suggest that combining DTI and GM volumetry may achieve better explanation of long-term motor deficit in stroke patients, than using either measure individually. This finding may provide guidance in determining optimal neurorehabilitative interventions.     

Chloroplast ATP synthase is reduced by both f-type and m-type thioredoxins

The activity of the molecular motor enzyme, chloroplast ATP synthase, is regulated in a redox-dependent manner. The γ subunit, CF₁-γ, is the central shaft of this enzyme complex and possesses the redox-active cysteine pair, which is reduced by thioredoxin (Trx). In light conditions, Trx transfers the reducing equivalent obtained from the photosynthetic electron transfer system to the CF₁-γ. Previous studies showed that the light-dependent reduction of CF₁-γ is more rapid than those of other Trx target proteins in the stroma. Although there are multiple Trx isoforms in chloroplasts, it is not well understood as to which chloroplast Trx isoform primarily contributes to the reduction of CF₁-γ, especially under physiological conditions. We therefore performed direct assessment of the CF₁-γ reduction capacity of each of the Trx isoforms. The kinetic analysis of the reduction process showed no significant difference in the reduction efficiency between two major chloroplast Trxs, namely Trx-f and Trx-m. Based on the thorough analyses of the CF₁-γ redox dynamics in Arabidopsis thaliana Trx mutant plants, we found that lack of Trx-f or Trx-m had no significant impact on the in vivo light-dependent reduction of CF₁-γ. The results showed that CF₁-γ can accept the reducing power from both Trx-f and Trx-m in chloroplasts.     

Characterization of the Mycobacterial AdnAB DNA Motor Provides Insights into the Evolution of Bacterial Motor-Nuclease Machines

Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k_cat 415 s⁻¹), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3′ → 5′ translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the “leading” AdnB and “lagging” AdnA motor domains track in tandem, 3′ to 5′, along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5′ and 3′ terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5′ strand, while the AdnB nuclease cleaves the displaced 3′ strand. We present evidence that the distinctive “molecular ruler” function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5′-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme.