Chemo-Mechanical Coupling in F1-ATPase Revealed by Catalytic Site Occupancy during Catalysis

F₁-ATPase is a rotary molecular motor in which the central γ subunit rotates inside a cylinder made of α₃β₃ subunits. To clarify how ATP hydrolysis in three catalytic sites cooperate to drive rotation, we measured the site occupancy, the number of catalytic sites occupied by a nucleotide, while assessing the hydrolysis activity under identical conditions. The results show hitherto unsettled timings of ADP and phosphate releases: starting with ATP binding to a catalytic site at an ATP-waiting γ angle defined as 0°, phosphate is released at ∼200°, and ADP is released during quick rotation between 240° and 320° that is initiated by binding of a third ATP. The site occupancy remains two except for a brief moment after the ATP binding, but the third vacant site can bind a medium nucleotide weakly.     

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.     

A change in the radius of rotation of F1-ATPase indicates a tilting motion of the central shaft

F₁-ATPase is a water-soluble portion of F_oF₁-ATP synthase and rotary molecular motor that exhibits reversibility in chemical reactions. The rotational motion of the shaft subunit γ has been carefully scrutinized in previous studies, but a tilting motion of the shaft has never been explicitly postulated. Here we found a change in the radius of rotation of the probe attached to the shaft subunit γ between two different intermediate states in ATP hydrolysis: one waiting for ATP binding, and the other waiting for ATP hydrolysis and/or subsequent product release. Analysis of this radial difference indicates a ∼4° outward tilting of the γ-subunit induced by ATP binding. The tilt angle is a new parameter, to our knowledge, representing the motion of the γ-subunit and provides a new constraint condition of the ATP-waiting conformation of F₁-ATPase, which has not been determined as an atomic structure from x-ray crystallography.     

Neither helix in the coiled coil region of the axle of F1-ATPase plays a significant role in torque production

F₁-ATPase is an ATP-driven rotary molecular motor in which the central γ-subunit rotates inside the cylinder made of α₃β₃ subunits. The amino and carboxy termini of the γ-subunit form the axle, an α-helical coiled coil that deeply penetrates the stator cylinder. We previously truncated the axle step by step, starting with the longer carboxy terminus and then cutting both termini at the same levels, resulting in a slower yet considerably powerful rotation. Here we examine the role of each helix by truncating only the carboxy terminus by 25-40 amino-acid residues. Longer truncation impaired the stability of the motor complex severely: 40 deletions failed to yield rotating the complex. Up to 36 deletions, however, the mutants produced an apparent torque at nearly half of the wild-type torque, independent of truncation length. Time-averaged rotary speeds were low because of load-dependent stumbling at 120° intervals, even with saturating ATP. Comparison with our previous work indicates that half the normal torque is produced at the orifice of the stator. The very tip of the carboxy terminus adds the other half, whereas neither helix in the middle of the axle contributes much to torque generation and the rapid progress of catalysis. None of the residues of the entire axle played a specific decisive role in rotation.     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

Theory of Site-Specific DNA-Protein Interactions in the Presence of Conformational Fluctuations of DNA Binding Domains

We develop a theory that explains how the thermally driven conformational fluctuations in the DNA binding domains (DBDs) of the DNA binding proteins (DBPs) are effectively coupled to the one-dimensional searching dynamics of DBPs for their cognate sites on DNA. We show that the rate γ_opt, associated with the flipping of conformational states of DBDs beyond which the maximum search efficiency of DBPs is achieved, varies with the one-dimensional sliding length L as γ_opt ∝ L⁻² and with the number of roadblock protein molecules present on the same DNA m as γ_opt ∝ m². The required free energy barrier E_RTO associated with this flipping transition seems to be varying with L as E_RTO ∝ ln L². When the barrier height associated with the conformational flipping of DBDs is comparable with that of the thermal free energy, then the possible value of L under in vivo conditions seems to be L ≤ 70 bps.     

Effect of external torque on the ATP-driven rotation of F1-ATPase

F₁-ATPase is a rotary molecular motor powered by the torque generated by another rotary motor F₀ to synthesize ATP in vivo. Therefore elucidation of the behavior of F₁ under external torque is very important. Here, we applied controlled external torque by electrorotation and investigated the ATP-driven rotation for the first time. The rotation was accelerated by assisting torque and decelerated by hindering torque, but F₁ rarely showed rotations in the ATP synthesis direction. This is consistent with the prediction by models based on the assumption that the rotation is tightly coupled to ATP hydrolysis and synthesis. At low ATP concentrations (2 and 5 μM), 120° stepwise rotation was observed. Due to the temperature rise during experiment, quantitative interpretation of the data is difficult, but we found that the apparent rate constant of ATP binding clearly decreased by hindering torque and increased by assisting torque.     

Effect of ε subunit on the rotation of thermophilic Bacillus F1-ATPase

F₁-ATPase is an ATP-driven motor in which γε rotates in the α₃β₃-cylinder. It is attenuated by MgADP inhibition and by the ε subunit in an inhibitory form. The non-inhibitory form of ε subunit of thermophilic Bacillus PS3 F₁-ATPase is stabilized by ATP-binding with micromolar K_d at 25 °C. Here, we show that at [ATP] > 2 μM, ε does not affect rotation of PS3 F₁-ATPase but, at 200 nM ATP, ε prolongs the pause of rotation caused by MgADP inhibition while the frequency of the pause is unchanged. It appears that ε undergoes reversible transition to the inhibitory form at [ATP] below K_d.     

None of the Rotor Residues of F1-ATPase Are Essential for Torque Generation

F₁-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor’s high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α₃β₃γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α₃β₃ subunits using the energy derived from ATP hydrolysis. The rotor consists of an axle, a coiled coil of the amino- and carboxyl-terminal α-helices of γ, which deeply penetrates the stator cylinder, and a globular protrusion that juts out from the stator. Previous work has shown that, for a thermophilic F₁, significant portions of the axle can be truncated and the motor still rotates a submicron sized bead duplex, indicating generation of up to half the wild-type (WT) torque. Here, we inquire if any specific interactions between the stator and the rest of the rotor are needed for the generation of a sizable torque. We truncated the protruding portion of the rotor and replaced part of the remaining axle residues such that every residue of the rotor has been deleted or replaced in this or previous truncation mutants. This protrusionless construct showed an unloaded rotary speed about a quarter of the WT, and generated one-third to one-half of the WT torque. No residue-specific interactions are needed for this much performance. F₁ is so designed that the basic rotor-stator interactions for torque generation and control of catalysis rely solely upon the shape and size of the rotor at very low resolution. Additional tailored interactions augment the torque to allow ATP synthesis under physiological conditions.     

Nucleotide-dependent shape changes in the reverse direction motor, myosin VI

We have studied the shape of myosin VI, the actin minus-end directed motor, by negative stain and metal shadow electron microscopy. Single particle processing was used to make two-dimensional averages of the stain images, which greatly increases the clarity and allows detailed comparisons with crystal structures. A total of 169,964 particle images were obtained from two different constructs in six different states (four nucleotide states and with and without Ca²⁺). The shape of truncated apo myosin VI was very similar to the apo crystal structure, with the lever arm bent strongly backward and around the motor domain. In the full-length molecule, the C-terminal part of the tail has an additional bend taking it back across the motor domain, which may reflect a regulated state. Addition of ATP, ADP, or ATP-γS resulted in a large change, straightening the molecule from the bent shape and swinging the lever by ∼140°. Although these nucleotides would not be expected to produce the pre-powerstroke state, myosin VI in their presence was most similar to the truncated crystal structure with bound ADP-VO₄, which is thought to show the pre-powerstroke shape. The nucleotide data were therefore substantially different from expectation based on crystal structures. The full-length molecule was almost completely monomeric; only ∼1% were dimers, joined through the ends of the tail. Addition of calcium ions appeared to result in release of the second calmodulin light chain. In negatively stained molecules there was little indication of extended α-helical structure in the tail, but molecules viewed by metal shadowing had a tail ∼3× longer, 29 vs. 9 nm, part of which is likely to be a single α-helix.     

Asymmetry of the GroEL-GroES complex under physiological conditions as revealed by small-angle x-ray scattering

Despite the well-known functional importance of GroEL-GroES complex formation during the chaperonin cycle, the stoichiometry of the complex has not been clarified. The complex can occur either as an asymmetric 1:1 GroEL-GroES complex or as a symmetric 1:2 GroEL-GroES complex, although it remains uncertain which type is predominant under physiological conditions. To resolve this question, we studied the structure of the GroEL-GroES complex under physiological conditions by small-angle x-ray scattering, which is a powerful technique to directly observe the structure of the protein complex in solution. We evaluated molecular structural parameters, the radius of gyration and the maximum dimension of the complex, from the x-ray scattering patterns under various nucleotide conditions (3 mM ADP, 3 mM ATPγS, and 3 mM ATP in 10 mM MgCl₂ and 100 mM KCl) at three different temperatures (10°C, 25°C, and 37°C). We then compared the experimentally observed scattering patterns with those calculated from the known x-ray crystallographic structures of the GroEL-GroES complex. The results clearly demonstrated that the asymmetric complex must be the major species stably present in solution under physiological conditions. On the other hand, in the presence of ATP (3 mM) and beryllium fluoride (10 mM NaF and 300 μM BeCl₂), we observed the formation of a stable symmetric complex, suggesting the existence of a transiently formed symmetric complex during the chaperonin cycle.     

Stoichiometric analysis and experimental investigation of glycerol–glucose co-fermentation in Klebsiella pneumoniae under microaerobic conditions

The ratio between two substrates is an important parameter in microbial co-fermentation, such as 1,3-propanediol production from glycerol by Klebsiella pneumoniae using glucose as the cosubstrate. In this study, the glycerol–glucose cometabolism by K. pneumoniae is stoichiometrically analyzed according to energy (ATP), reducing equivalent (NADH₂) and product balances. The theoretical analysis reveals that the yield of 1,3-propanediol to glycerol under microaerobic conditions depends not only on the ratio of glucose to glycerol initially added, but also on the molar fraction of reducing equivalent oxidized completely by molecular oxygen in tricarboxylic acid (TCA) cycle (δ) and the molar fraction of TCA cycle in acetyl-CoA metabolism (γ). The maximum ratio of 0.32 mol glucose per mol glycerol is needed to convert glycerol completely to 1,3-propanediol under anaerobic conditions if glycerol neither enters oxidation pathways nor forms biomass. The ratio can be reduced under microaerobic conditions. The experimental results of batch cultures demonstrate that the biomass concentration and yield of 1,3-propanediol on glycerol could be enhanced by using glucose as a co-substrate. The theoretical analysis reveals the relationship between yield of 1,3-propanediol to glycerol, ratio of glucose to glycerol and respiratory quotient (RQ). These results are helpful for the experimental design and control.     

Larsenia salina gen. nov., sp. nov., a new member of the family Halomonadaceae based on multilocus sequence analysis

Two Gram-staining-negative, moderately halophilic bacteria, strains M1-18^T and L1-16, were isolated from a saltern located in Huelva (Spain). They were motile, strictly aerobic rods, growing in the presence of 3–25% (w/v) NaCl (optimal growth at 7.5–10% [w/v] NaCl), between pH 4.0 and 9.0 (optimal at pH 6.0–7.0) and at temperatures between 15 and 40 °C (optimal at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed the higher similarity values with Chromohalobacter israelensis ATCC 43985^T (95.2–94.8%) and Chromohalobacter salexigens DSM 3043^T (95.0–94.9%), and similarity values lower than 94.6% with other species of the genera Chromohalobacter, Kushneria, Cobetia or Halomonas. Multilocus sequence analysis (MLSA) based on the partial sequences of atpA, rpoD and secA housekeeping genes indicated that the new isolates formed an independent and monophyletic branch that was related to the peripheral genera of the family Halomonadaceae, Halotalea, Carnimonas and Zymobacter, supporting their placement as a new genus of the Halomonadaceae. The DNA–DNA hybridization between both strains was 82%, whereas the values between strain M1-18^T and the most closely related species of Chromohalobacter and Kushneria were equal or lower to 48%. The major cellular fatty acids were C_18:1ω7c/C_18:1ω6c, C_16:0, and C_16:1ω7c/C_16:1ω6c, a profile that differentiate this new taxon from species of the related genera. We propose the placement of both strains as a novel genus and species, within the family Halomonadaceae, with the name Larsenia salina gen. nov., sp. nov. The type strain is M1-18^T (= CCM 8464 = CECT 8192^T = IBRC-M 10767^T = LMG 27461^T).     

Adsorption of Victoria blue by carbon/Ba/alginate beads: Kinetics,thermodynamics and isotherm studies

The kinetic, thermodynamic and isotherm modeling studies were carried out on adsorptive removal of Victoria blue (VB) dye using activated carbon, Ba/alginate and modified carbon/Ba/alginate polymer beads. The feasibility of sorption process was determined by varying the experimental parameters viz., dye concentration (4–20 mg L⁻¹), contact time (10–90 min), pH (3–10), adsorbent dose (0.5–2.5 g) and temperature (303–343 K). Freundlich and Langmuir isotherms were determined and ascertained with the dimensionless separation factor (R_L). Lagergren's pseudo-first order, pseudo-second order and intraparticle diffusion model equations were used to analyze the kinetics of the adsorption process. The thermodynamic consistency of adsorption was found with Gibbs free energy (ΔG°), changes in enthalpy (ΔH°) and entropy (ΔS°) were calculated using the Van’t Hoff plot. The polymer beads were characterized using Fourier Transform Infrared Spectroscopy (FTIR) and their morphology was determined by scanning electron microscopy (SEM).     

The expression of the skeletal muscle force-length relationship in vivo: A simulation study

The force-length relationship is one of the most important mechanical characteristics of skeletal muscle in humans and animals. For a physiologically realistic joint range of motion and therefore range of muscle fibre lengths only part of the force-length curve may be used in vivo, i.e. only a section of the force-length curve is expressed. A generalised model of a mono-articular muscle-tendon complex was used to examine the effect of various muscle architecture parameters on the expressed section of the force-length relationship for a 90° joint range of motion. The parameters investigated were: the ratio of tendon resting length to muscle fibre optimum length (L_TR:L_F·OPT) (varied from 0.5 to 11.5), the ratio of muscle fibre optimum length to average moment arm (L_F·OPT:r) (varied from 0.5 to 5), the normalised tendon strain at maximum isometric force (c) (varied from 0 to 0.08), the muscle fibre pennation angle (θ) (varied from 0° to 45°) and the joint angle at which the optimum muscle fibre length occurred (φ). The range of values chosen for each parameter was based on values reported in the literature for five human mono-articular muscles with different functional roles. The ratios L_TR:L_F·OPT and L_F·OPT:r were important in determining the amount of variability in the expressed section of the force-length relationship. The modelled muscle operated over only one limb at intermediate values of these two ratios (L_TR:L_F·OPT=5; L_F·OPT:r=3), whether this was the ascending or descending limb was determined by the precise values of the other parameters. It was concluded that inter-individual variability in the expressed section of the force-length relationship is possible, particularly for muscles with intermediate values of L_TR:L_F·OPT and L_F·OPT:r such as the brachialis and vastus lateralis. Understanding the potential for inter-individual variability in the expressed section is important when using muscle models to simulate movement.     

Water transport in epididymal and ejaculated rhesus monkey (Macaca mulatta) sperm during freezing

In the present study, we report the effects of cooling ejaculated and epididymal rhesus monkey (Macacamulatta) sperm with and without the presence of a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal sperm cell suspensions were obtained at a cooling rate of 20 °C/min in the absence of any cryoprotective agents and in the presence of 0.7 M of glycerol, as well. Using previously published values, the macaque sperm cell was modeled as a cylinder of length 73.83 μm with a radius of 0.40 μm and an osmotically inactive cell volume, V_b, of 0.772V_o, where V_o is the isotonic cell volume. This translated to a surface area, SA to initial water volume, WV ratio of ∼22 μm⁻¹. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water at 0 °C, L_pg or L_pg[cpa] and the activation energy, E_Lp or E_Lp[cpa]) were found to range from: L_pg or L_pg[cpa] = 0.0020-0.0029 μm/min-atm; E_Lp or E_Lp[cpa]) = 10.6-18.3 kcal/mole. By incorporating these membrane permeability parameters in a recently developed equation (optimal cooling rate, ; where the units of B_opt are °C/min, E_Lp or E_Lp[cpa] are kcal/mole, L_pg or L_pg[cpa] are μm/min-atm and SA/WV are μm⁻¹), we determined the optimal rates of freezing macaque sperm to be ∼23 °C/min (ejaculated sperm in the absence of CPAs), ∼29 °C/min (ejaculated sperm in the presence of glycerol), ∼24 °C/min (epididymal sperm in the absence of CPAs) and ∼24 °C/min (epididymal sperm in the presence of glycerol). In conclusion, the subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal macaque spermatozoa under corresponding cooling conditions.     

Osmotic parameters of red blood cells from umbilical cord blood

The transfusion of red blood cells from umbilical cord blood (cord RBCs) is gathering significant interest for the treatment of fetal and neonatal anemia, due to its high content of fetal hemoglobin as well as numerous other potential benefits to fetuses and neonates. However, in order to establish a stable supply of cord RBCs for clinical use, a cryopreservation method must be developed. This, in turn, requires knowledge of the osmotic parameters of cord RBCs. Thus, the objective of this study was to characterize the osmotic parameters of cord RBCs: osmotically inactive fraction (b), hydraulic conductivity (L_p), permeability to cryoprotectant glycerol (P_glycerol), and corresponding Arrhenius activation energies (E_a). For L_p and P_glycerol determination, RBCs were analyzed using a stopped-flow system to monitor osmotically-induced RBC volume changes via intrinsic RBC hemoglobin fluorescence. L_p and P_glycerol were characterized at 4 °C, 20 °C, and 35 °C using Jacobs and Stewart equations with the E_a calculated from the Arrhenius plot. Results indicate that cord RBCs have a larger osmotically inactive fraction compared to adult RBCs. Hydraulic conductivity and osmotic permeability to glycerol of cord RBCs differed compared to those of adult RBCs with the differences dependent on experimental conditions, such as temperature and osmolality. Compared to adult RBCs, cord RBCs had a higher E_a for L_p and a lower E_a for P_glycerol. This information regarding osmotic parameters will be used in future work to develop a protocol for cryopreserving cord RBCs.     

Remarkable stability of the proton translocating F1FO-ATP synthase from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

For functional characterization, we isolated the F₁F_O-ATP synthase of the thermophilic cyanobacterium Thermosynechococcus elongatus. Because of the high content of phycobilisomes, a combination of dye-ligand chromatography and anion exchange chromatography was necessary to yield highly pure ATP synthase. All nine single F₁F_O subunits were identified by mass spectrometry. Western blotting revealed the SDS stable oligomer of subunits c in T. elongatus. In contrast to the mass archived in the database (10,141 Da), MALDI-TOF-MS revealed a mass of the subunit c monomer of only 8238 Da. A notable feature of the ATP synthase was its ability to synthesize ATP in a wide temperature range and its stability against chaotropic reagents. After reconstitution of F₁F_O into liposomes, ATP synthesis energized by an applied electrochemical proton gradient demonstrated functional integrity. The highest ATP synthesis rate was determined at the natural growth temperature of 55 °C, but even at 95 °C ATP production occurred. In contrast to other prokaryotic and eukaryotic ATP synthases which can be disassembled with Coomassie dye into the membrane integral and the hydrophilic part, the F₁F_O-ATP synthase possessed a particular stability. Also with the chaotropic reagents sodium bromide and guanidine thiocyanate, significantly harsher conditions were required for disassembly of the thermophilic ATP synthase.     

On chemomechanical coupling of the F1-ATPase molecular motor

F₁-ATPase catalyzes ATP hydrolysis to drive the central γ-shaft rotating inside a hexameric cylinder composed of alternating α and β subunits. Experiments showed that the rotation of γ-shaft proceeds in steps of 120° and each 120°-rotation is composed of an 80° substep and a 40° substep. Here, based on the previously proposed models, an improved physical model for chemomechanical coupling of F₁-ATPase is presented, with which the two-substep rotation is well explained. One substep is driven by the power stroke upon ATP binding, while the other one resulted from the passage of γ-shaft from previous to next adjacent β subunits via free diffusion. Using the model, the dynamics and kinetics of F₁-ATPase, such as the rotating time of each substep, the dwell time at each pause and the rotation rate, are analytically studied. The theoretical results obtained with only three adjustable parameters reproduce the available experimental data well.