Various Concentrations Of ATP Research Articles

Timing and Load-dependence of the Powerstroke and Pi-release in Skeletal Muscle Myosin

Muscle myosin converts chemical energy from ATP into mechanical work to power contraction, however it is still unclear how this transduction occurs at the molecular level. A key unanswered question is whether phosphate release precedes the powerstroke or if the powerstroke occurs first. This has been a challenging question to answer because these events occur faster than the time resolution of most instruments. Therefore, we used an ultrafast single molecule laser trap assay, with sub-millisecond time resolution, to directly visualize the timing of the powerstorke in fast skeletal muscle myosin. By elevating the level of phosphate in the buffer (15 mM) under varying ATP concentration, we ensured that myosin remained in a phosphate (Pi) bound state. We observed that myosin generated a powerstroke in the presence of phosphate that was similar in size (∼5 nm) and rate (∼5000 s−1 at 3 pN resistive force) to that observed in the absence of phosphate. This suggests that myosin generates its powerstroke prior to the release of phosphate. Further analyses are under way to determine if elevated levels of phosphate induced reversal of the powerstroke later in the binding event. Additional experiments were done over a range of assistive and resistive loads, from −6pN to +6pN, to determine the load-dependence of the powerstroke and the effect on myosin detachment rate from different chemomechanical states. Thus these data provide novel insights into the fundamental nature of force generation by muscle myosin.

Calcium binding to a remote site can replace magnesium as cofactor for mitochondrial Hsp90 (TRAP1) ATPase activity

The Hsp90 molecular chaperones are ATP-dependent enzymes that maintain protein homeostasis and regulate many essential cellular processes. Higher eukaryotes have organelle-specific Hsp90 paralogs that are adapted to each subcellular environment. The mitochondrial Hsp90, TNF receptor–associated protein 1 (TRAP1), supports the folding and activity of electron transport components and is increasingly appreciated as a critical player in mitochondrial signaling. Calcium plays a well-known and important regulatory role in mitochondria where it can accumulate to much higher concentrations than in the cytoplasm. Surprisingly, we found here that calcium can replace magnesium, the essential enzymatic cofactor, to support TRAP1 ATPase activity. Anomalous X-ray diffraction experiments revealed a calcium-binding site within the TRAP1 nucleotide-binding pocket located near the ATP α-phosphate and completely distinct from the magnesium-binding site adjacent to the β- and γ-phosphates. In the presence of magnesium, ATP hydrolysis by TRAP1, as with other Hsp90s, was noncooperative, whereas calcium binding resulted in cooperative hydrolysis by the two protomers within the Hsp90 dimer. The structural data suggested a mechanism for this cooperative behavior. Because of the cooperativity, at high ATP concentrations, ATPase activity was higher with calcium, whereas the converse was observed at low ATP concentrations. Integrating these observations, we propose a model in which the divalent cation choice can control switching between noncooperative and cooperative TRAP1 ATPase mechanisms in response to varying ATP concentrations. This switching may facilitate coordination between cellular energetics, mitochondrial signaling, and protein homeostasis via alterations in the TRAP1 ATP-driven cycle and its consequent effects on different mitochondrial clients.

The Effects of the Interplay between Motor and Brownian Forces on the Rheology of Active Gels.

Active gels perform key mechanical roles inside the cell, such as cell division, motion, and force sensing. The unique mechanical properties required to perform such functions arise from the interactions between molecular motors and semiflexible polymeric filaments. Molecular motors can convert the energy released in the hydrolysis of ATP into forces of up to piconewton magnitudes. Moreover, the polymeric filaments that form active gels are flexible enough to respond to Brownian forces but also stiff enough to support the large tensions induced by the motor-generated forces. Brownian forces are expected to have a significant effect especially at motor activities at which stable noncontractile in vitro active gels are prepared for rheological measurements. Here, a microscopic mean-field theory of active gels originally formulated in the limit of motor-dominated dynamics is extended to include Brownian forces. In the model presented here, Brownian forces are included accurately, at real room temperature, even in systems with high motor activity. It is shown that a subtle interplay, or competition, between motor-generated forces and Brownian forces has an important impact on the mass transport and rheological properties of active gels. The model predictions show that at low frequencies the dynamic modulus of active gels is determined mostly by motor protein dynamics. However, Brownian forces significantly increase the breadth of the relaxation spectrum and can affect the shape of the dynamic modulus over a wide frequency range even for ratios of motor to Brownian forces of more than a hundred. Since the ratio between motor and Brownian forces is sensitive to ATP concentration, the results presented here shed some light on how the transient mechanical response of active gels changes with varying ATP concentration.

Ionotropic Purinergic Receptors P2X4 and P2X7: Proviral or Antiviral? An Insight into P2X Receptor Signaling and Hepatitis C Virus Infection.

Purinergic P2X receptors are plasma membrane bound, ATP-gated ion channels that are expressed on wide range of cells and respond to varying ATP concentrations in extracellular environment. Upon activation they increase membrane permeability for Ca(2+) ions and trigger a cascade of signaling complexes. During the course of hepatitis C virus (HCV) infection, ATP is released from the infected hepatocyte, which binds with Purinergic receptors (P2X) on peripheral blood mononuclear cells (PBMCs) and initiate downstream signaling pathways by disturbing the ionic balance of the cell. The present study investigates quantitative expression of P2X7 and P2X4 along with selected host genes PEPCK, transforming growth factor β (TGF-β), MAPK, Rho, and Akt in PBMCs of chronic HCV infection patients. PBMCs were isolated from collected blood samples of study subjects. Transcript analysis of P2X7, P2X4, and targeted downstream genes was done using quantitative real-time polymerase chain reaction. Relative expression analysis was performed by unpaired Student's t test on GraphPad Prism version 5. We found a notable increase of threefolds and 1.8-folds in the expression of P2X7 and P2X4 receptors in treatment naïve category while the expression of PEPCK, TGF-β, MAPK, AKT, and Rho A increased by 2.8, 1.9, 2.2, 2.2, and 1.8-folds, respectively. In sustained virological response patients, P2X7 significantly increased up to 3.5-folds while the expression of P2X4 receptor was increased up to twofold. In third category, treatment nonresponder, the expression of P2X7, P2X4 receptors, and targeted markers remained un-altered. This study deals with two major aspects of P2X4 and P2X7 receptors in PBMCs of chronic HCV individuals. One is their role in providing antiviral immunity to host against HCV; second aspect is the role of P2X receptors in inducing HCV pathogenesis via AKT, TGF-β, Rho A, PEPCK, and MAPK expression.

Structural Basis for Nucleotide Hydrolysis by the Acid Sphingomyelinase-like Phosphodiesterase SMPDL3A

Sphingomyelin phosphodiesterase, acid-like 3A (SMPDL3A) is a member of a small family of proteins founded by the well characterized lysosomal enzyme, acid sphingomyelinase (ASMase). ASMase converts sphingomyelin into the signaling lipid, ceramide. It was recently discovered that, in contrast to ASMase, SMPDL3A is inactive against sphingomyelin and, surprisingly, can instead hydrolyze nucleoside diphosphates and triphosphates, which may play a role in purinergic signaling. As none of the ASMase-like proteins has been structurally characterized to date, the molecular basis for their substrate preferences is unknown. Here we report crystal structures of murine SMPDL3A, which represent the first structures of an ASMase-like protein. The catalytic domain consists of a central mixed β-sandwich surrounded by α-helices. Additionally, SMPDL3A possesses a unique C-terminal domain formed from a cluster of four α-helices that appears to distinguish this protein family from other phosphoesterases. We show that SMDPL3A is a di-zinc-dependent enzyme with an active site configuration that suggests a mechanism of phosphodiester hydrolysis by a metal-activated water molecule and protonation of the leaving group by a histidine residue. Co-crystal structures of SMPDL3A with AMP and α,β-methylene ADP (AMPCP) reveal that the substrate binding site accommodates nucleotides by establishing interactions with their base, sugar, and phosphate moieties, with the latter the major contributor to binding affinity. Our study provides the structural basis for SMPDL3A substrate specificity and sheds new light on the function of ASMase-like proteins.

Do cardiac actin mutations lead to altered actomyosin interactions?

It is currently hypothesized that increased heart muscle contractility leads to hypertrophic cardiomyopathy (HCM), and reduced contractility leads to dilated cardiomyopathy (DCM). To determine if changes in the core interaction between actin and myosin occur due to mutations in the cardiac actin gene (ACTC), we measured the interactions between myosin and 8 ACTC mutant proteins found in patients with HCM or DCM. R312H showed a decreased actin-activated myosin S1 ATPase rate (13.1 ± 0.63 μmol/L/min) compared to WT (15.3 ± 1.6 μmol/L/min), whereas the rate with E99K was significantly higher (20.1 ± 1.5 μmol/L/min). In vitro motility assays with varying ATP concentrations showed that the KM for E99K remains unchanged with a significantly decreased Vmax (1.90 ± 0.37 μm/sec) compared to WT (3.33 ± 0.46 μm/sec). Based on a 5 nm myosin step size, we calculated a duty ratio of approximately 0.04 for WT and the majority of mutant actins; however, the duty ratio for E99K was twice as high. Based on our analysis of 8 ACTC mutants, we infer that mutations in ACTC lead to disease through various molecular mechanisms. While changes in actomyosin interactions with the E99K mutation might cause increased ATP usage and tension leading to HCM, measurable changes in the basic interaction between actin and myosin do not appear to be involved in the mechanisms of disease development for the other ACTC mutants tested.

Do Cardiac Actin Mutations Lead to Altered Muscle Contractility?

The hypothesis that increased heart muscle contractility leads to HCM and reduced contractility leads to DCM is based mainly on studies on myosin motor proteins. This hypothesis predicts that a change in cardiac actin (ACTC) may impact muscle contractility. To test this prediction, we determined the interactions between myosin and 8 of 16 ACTC mutant proteins that are known to cause hypertrophic or dilated cardiomyopathy. R312H showed a decreased actin-activated S1 ATPase rate (13.1±0.63 μM/min) compared to WT (15.3±1.58 μM/min), whereas the rate with E99K was significantly higher (20.1±1.46μM/min). In vitro motility assays were performed with varying ATP concentrations. E99K exhibited increased KM (0.086±0.02 μM) compared to WT (0.068±0.02 μM). E99K demonstrated a significantly decreased Vmax (1.93±0.1 μm/sec) compared to WT (3.31±0.12 μm/sec). In contrast, M305 had a similar KM (0.088±0.01 μm/sec) to E99K, but its Vmax (3.39±0.11μm/sec) was similar to WT. Based on a 5 nm myosin step size, we calculated a duty ratio of about 0.04 for WT and most mutant actins; however, the duty ratio was twice as high for E99K. With thin-filament extracted and reconstituted muscle fibers with E99K, a slight, but significant increase in Ca2+sensitivity (ΔpCa=0.11±0.04) was observed; consequently tension was slightly larger than that of WT for pCa ranging 5.6-6.0, without changing maximum tension at pCa 4.66. These experiments suggest that the primary change with the E99K actin mutant is enhanced ATP usage and increased tension at partial activation, which lead to HCM.

Single-Molecule Measurements to Investigate the Negative Cooperativity in Na+/K+ -ATPase

The Na+/K+-ATPase, a cell membrane ion motive ATPase, uses energy from the hydrolysis of ATP to move Na+ out of and K+ into cells, thus maintaining the membrane resting potential and cellular volume. To investigate how this pump functions, we isolated ATPase from duck supraorbital salt glands and labeled it with Cy3-maleimide (Cy3-ATPase). In bulk experiments, we found that the fluorescence of Cy3-ATPase decreases in the presence of ATP (Biochim Biophys Acta 2009; 1794:1549-1557). The kinetics of this ATP-induced fluorescence decrease exhibited negative cooperativity and could be explained in terms of protein aggregation. To further explore the phenomenon of negative cooperativity on the level of individual monomers, we used single-molecule total internal reflection fluorescence (SM-TIRF) microscopy. Protein monomers were solubilized and reconstituted into lipid vesicles to investigate the effect of varying ATP concentration on the fluorescence.Data from SM-TIRF experiments, analyzed using a hidden Markov model (HMM), suggest that the Cy3-ATPase exists in dynamic equilibrium between a high fluorescence state (unquenched) and a low fluorescence state (partially quenched). These kinetics are characterized by either rapid or slow transitions between these states. Two subpopulations are observed, one where the transitions between the states occur rapidly and the other where the kinetics are slower. Preliminary analysis of the data suggests that ATP shifts the population distribution from those exhibiting rapid transitions to those exhibiting slow transitions. Here, we report on the analysis of these effects and the implications of the above observations on the working of the pump.

Basic Properties of Rotary Dynamics of the Molecular Motor Enterococcus hirae V1-ATPase

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe. EhV1 rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (Vmax) of 107 revolutions/s and a Michaelis constant (Km) of 154 μM at 26 °C. At all ATP concentrations tested, EhV1 showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V1-ATPase (TtV1). At 10 μM ATP (<<Km), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 × 10(6) M(-1) s(-1). At 40 mM ATP (>>Km), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV1. Our results identify the common properties of rotary catalysis of V1-ATPases that are distinct from those of F1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.

Regulation of Cardiac ATP-sensitive Potassium Channel Surface Expression by Calcium/Calmodulin-dependent Protein Kinase II

Cardiac ATP-sensitive potassium (K(ATP)) channels are key sensors and effectors of the metabolic status of cardiomyocytes. Alteration in their expression impacts their effectiveness in maintaining cellular energy homeostasis and resistance to injury. We sought to determine how activation of calcium/calmodulin-dependent protein kinase II (CaMKII), a central regulator of calcium signaling, translates into reduced membrane expression and current capacity of cardiac K(ATP) channels. We used real-time monitoring of K(ATP) channel current density, immunohistochemistry, and biotinylation studies in isolated hearts and cardiomyocytes from wild-type and transgenic mice as well as HEK cells expressing wild-type and mutant K(ATP) channel subunits to track the dynamics of K(ATP) channel surface expression. Results showed that activation of CaMKII triggered dynamin-dependent internalization of K(ATP) channels. This process required phosphorylation of threonine at 180 and 224 and an intact (330)YSKF(333) endocytosis motif of the K(ATP) channel Kir6.2 pore-forming subunit. A molecular model of the μ2 subunit of the endocytosis adaptor protein, AP2, complexed with Kir6.2 predicted that μ2 docks by interaction with (330)YSKF(333) and Thr-180 on one and Thr-224 on the adjacent Kir6.2 subunit. Phosphorylation of Thr-180 and Thr-224 would favor interactions with the corresponding arginine- and lysine-rich loops on μ2. We concluded that calcium-dependent activation of CaMKII results in phosphorylation of Kir6.2, which promotes endocytosis of cardiac K(ATP) channel subunits. This mechanism couples the surface expression of cardiac K(ATP) channels with calcium signaling and reveals new targets to improve cardiac energy efficiency and stress resistance.

The Relationship of Motor Attachment Geometry and Velocity Fluctuations in Kinesin-Microtubule Gliding Motility Assays

In gliding motility assays, surface-adhered motor proteins bind to the gliding filaments in a variety of orientations. The resulting variations in the angle of attachment affect the efficiency of force transfer onto the filament, which in turn results in fluctuations of the gliding velocity as shown theoretically by Sekimoto and Tawada [1]. The theory establishes a relationship between the motional diffusion coefficient, the gliding velocity, the motor density, and a factor capturing the angular variations in the motor attachment. We present measurements of the motional diffusion coefficient for microtubules gliding on full-length kinesin-1 with known surface density. Measurements at varying ATP concentrations and kinesin surface densities enable, for the first time, measurements of the geometry factor. The implications of these measurements for the underlying motor attachment geometry as well as the efficiency of force generation by coupled motors will be discussed.1. Sekimoto, K.; Tawada, K. Phys Rev Lett 1995, 75, (1), 180-183.

Relationships between Free-Living Protozoa, Cultivable Legionella spp., and Water Quality Characteristics in Three Drinking Water Supplies in the Caribbean

The study whose results are presented here aimed at identifying free-living protozoa (FLP) and conditions favoring the growth of these organisms and cultivable Legionella spp. in drinking water supplies in a tropical region. Treated and distributed water (±30°C) of the water supplies of three Caribbean islands were sampled and investigated with molecular techniques, based on the 18S rRNA gene. The protozoan host Hartmannella vermiformis and cultivable Legionella pneumophila were observed in all three supplies. Operational taxonomic units (OTUs) with the highest similarity to the potential or candidate hosts Acanthamoeba spp., Echinamoeba exundans, E. thermarum, and an Neoparamoeba sp. were detected as well. In total, 59 OTUs of FLP were identified. The estimated protozoan richness did not differ significantly between the three supplies. In supply CA-1, the concentration of H. vermiformis correlated with the concentration of Legionella spp. and clones related to Amoebozoa predominated (82%) in the protozoan community. These observations, the low turbidity (<0.2 nephelometric turbidity units [NTU]), and the varying ATP concentrations (1 to 12 ng liter(-1)) suggest that biofilms promoted protozoan growth in this supply. Ciliophora represented 25% of the protozoan OTUs in supply CA-2 with elevated ATP concentrations (maximum, 55 ng liter(-1)) correlating with turbidity (maximum, 62 NTU) caused by corroding iron pipes. Cercozoan types represented 70% of the protozoan clones in supply CA-3 with ATP concentrations of <1 ng liter(-1) and turbidity of <0.5 NTU in most samples of distributed water. The absence of H. vermiformis in most samples from supply CA-3 suggests that growth of this protozoan is limited at ATP concentrations of <1 ng liter(-1).

Purification and functional analysis of protein kinase G-1α using a bacterial expression system

3′,5′ Cyclic guanosine monophosphate (cGMP)-dependent protein kinase G-1α (PKG-1α) is an enzyme that is a target of several anti-hypertensive and erectile dysfunction drugs. Binding of cGMP to PKG-1α produces a conformational change that leads to enzyme activation. Activated PKG-1α performs important roles both in blood vessel vasodilation and in maintaining the smooth muscle cell in a differentiated contractile state. Recombinant PKG-1α has been expressed and purified using Sf9-insect cells. However, attempts at purifying full length protein in a soluble and active form in prokaryotes have thus far been unsuccessful. These attempts have been hampered by the lack of proper eukaryotic protein folding machinery in bacteria. In this study, we report the successful expression and purification of PKG-1α using a genetically engineered Escherichia coli strain, Rosetta-gami 2(DE3), transduced with full-length human PKG-1α cDNA containing a C-terminal histidine tag. PKG-1α was purified to homogeneity using sequential nickel affinity chromatography, gel filtration and ion exchange MonoQ columns. Protein identity was confirmed by immunoblot analysis. N-terminal sequencing using Edman degradation demonstrated that the purified protein was full length. Analysis of enzyme kinetics, using a nonlinear regression curve, identified that, at constant cGMP levels (10μM) and varying ATP concentrations, PKG-1α had a maximal velocity (Vmax) of 5.02±0.25pmol/min/μg and a Michaelis–Menten constant (Km) of 11.78±2.68μM ATP. Recent studies have suggested that endothelial function can be attenuated by oxidative and/or nitrosative stress but the role of PKG-1α under these conditions is unclear. We found that PKG-1α enzyme activity was attenuated by exposure to the NO donor, spermine NONOate, hydrogen peroxide, and peroxynitrite but not by superoxide, suggesting that the attenuation of PKG-1α activity may be an under-appreciated mechanism underlying the development of endothelial dysfunction in a number of cardiovascular diseases.

Kinetic Characterization of Non-Muscle Myosin IIB Single-Headed Heavy Meromyosin on Single Molecule Level with Optical Tweezers

Non-muscle myosin IIB (NMIIB) is a cytoplasmic conventional myosin, which plays an important role in development of the brain and heart, and in directed growth cone motility by maintaining cortical tension in motile cells. It forms short bipolar filaments with ∼14 myosin molecules on each side of the bare zone. NMIIB is a very slow myosin both in terms of actin-activated ATPase activity and actin translocation capability. Our previous studies showed that the NMIIB is a moderately high duty ratio (at least 20-25%) motor. The ADP release step (∼0.35 s-1), of NMIIB is only ∼3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s-1). Because of its slow ADP off-rate, acto-NMIIB has the highest ADP-affinity reported so far for the myosin superfamily (<0.15 μM). To examine the mechanics and kinetics of NMIIB at the single-molecule level we used a dual-beam optical tweezer to perform the three-bead assay. The surface-immobilized bead was coated with recombinantly engineered single-headed heavy meromyosin-like (NMIIB-SH-HMM) molecules. We measured the lifetimes of unitary actomyosin interactions and determined the actin-detachment kinetics with varying ATP concentrations. Results showed that at physiological ATP concentration (1 mM), the rate of detachment of acto-NMIIB-SH-HMM interactions was ∼0.51 s-1, similar to the ADP release rate and steady-state ATPase rate reported from solution kinetic studies. Decreasing the ATP concentration to 1 μM did not alter this rate of detachment (∼0.47 s-1). Also, our results showed that the power-stroke of NMIIB-SH-HMM was ∼8 nm. We will discuss our single-molecule results from the perspective of the essential cellular functions of NMIIB in cell locomotion, tension generation and maintenance.

Mechanism of Inhibition by C-terminal α-Helices of the ϵ Subunit of Escherichia coli FoF1-ATP Synthase

The epsilon subunit of bacterial FoF1-ATP synthase (FoF1), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal alpha-helices of epsilon, and the "extended" but not "hairpin-folded" state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of epsilon has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli FoF1 (EFoF1) mutant in which the epsilon subunit lacked the C-terminal domain (FoF1epsilonDeltaC), and ATP synthesis driven by acid-base transition (DeltapH) and the K+-valinomycin diffusion potential (DeltaPsi) was compared in detail with that of the wild-type enzyme (FoF1epsilonWT). The turnover numbers (kcat) of FoF1epsilonWT were severalfold lower than those of FoF1epsilonDeltaC. FoF1epsilonWT showed higher Michaelis constants (Km). The dependence of the activities of FoF1epsilonWT and FoF1epsilonDeltaC on various combinations of DeltapH and DeltaPsi was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of epsilon. Solubilized FoF1epsilonWT also showed lower kcat and higher Km values for ATP hydrolysis than the corresponding values of FoF1epsilonDeltaC. These results suggest that the C-terminal domain of the epsilon subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the gamma subunit.

The Mechanical Properties of Drosophila Jump Muscle Expressing Wild-type and an Embryonic Myosin Isoform

Transgenic Drosophila are highly useful for muscle protein structure-function studies, particularly myosin isoform diversity. However, our ability to mechanically analyze mutant proteins in Drosophila muscle has been limited to the skinned indirect flight muscle (IFM) preparation. We have developed a new preparation using the Drosophila tergal depressor of trochanter muscle (TDT) that increases our experiments to include maximum shortening velocity (Vmax), force-velocity relations, and steady-state power generation, which are not possible using IFM fibers. As with the IFM, we can replace the native TDT myosin with our myosin of choice. When expressing its native isoform (P2), the TDT is equivalent to a very fast vertebrate muscle, with a Vmax of 6.1±0.3 muscle lengths/second at 15°C, a steep tension-pCa curve, a Hill coefficient of 11±2, a high active isometric tension of 37±3 mN/mm2, and maximum power production (Pmax) at 43% of Vmax and 42% of maximum tension. Expressing an embryonic myosin isoform (EMB) in the TDT muscle decreased Vmax, isometric tension and Pmax by 50%, and the tension-pCa Hill coefficient decreased to 6±2. Varying ATP concentration, while measuring Vmax, revealed a higher ATP affinity for EMB than P2. Increasing Pi concentration reduced isometric tension of TDT expressing either isoform. A slight decrease in TDT Vmax with increasing Pi concentration suggests TDT Vmax may be influenced by Pi release rate. TDT Vmax was not influenced by [Pi] when expressing EMB. With our advances in the TDT preparation we will now be able to test a wider speed range of myosin isoforms, including the superfast IFM myosin, to test our hypothesis that a step associated with Pi release is rate limiting for Vmax of very fast myosins, while a step associated with ADP release is limiting for slower isoforms.

Disrupting Myosin Relay-Converter Domain Communication Impairs Drosophila Muscle Mechanical Performance and Flight Ability

We evaluated the mechanical properties of Drosophila indirect flight muscle (IFM) fibers expressing a myosin converter domain R759E mutation. The interaction of R759 with relay loop residue I508 is thought to be critical for relay-converter inter domain communication. By changing the charge on residue 759, we are testing if this inter domain interaction is important for the mechanical performance of muscle fibers. Electron microscopic examination of muscle fibers from young adult R759E flies indicates normal myofibril assembly. Using the work loop analysis technique we found that the maximum power (Pmax) generated by the mutant R759E fibers from two day old flies was significantly reduced by 50% compared to control fibers while the frequency at which maximum power is generated (fmax) was reduced to 67%. Maximum power occurred at peak-to-peak strain amplitude of 2% resting muscle fiber length. Varying ATP concentration at 15°C revealed no significant difference in Km for Pmax or fmax between control and mutant R759E fibers, suggesting that the mutation does not affect ATP affinity. Small amplitude sinusoidal analysis revealed a significant reduction in complex stiffness by 48% compared to control fibers, with elastic modulus, Ee, reduced by 31% and viscous modulus, Ev, reduced by 45%. This reduction in power and mechanical performance of the flight muscle fibers led to a decrease in wing beat frequency from 140 ± 2 Hz for control flies to 127 ± 2 Hz. The reduction in wing beat frequency contributed to a decrease in flight index from 2.31 ± 0.1 for control flies to 1.25 ± 0.1 at 15°C. Thus, this study suggests that the interaction between relay loop I508 and converter domain R759 is critical for myosin inter domain communication, muscle fiber power generation and Drosophila flight performance.

A Kinetic Model of Coordinated Myosin V

We present a kinetic model for the walking of myosin V on actin under conditions of zero external force. The model includes three pathways and the termination of the processivity. Experimentally measured kinetic parameters are used in the model to obtain quantitative results. Using the model and associated parameters, we compute the proportion of the pathway containing an intermediate state, as well as the walking velocities and run lengths at various concentrations of ATP and ADP. The resulting trends agree with experimental data. The model explains the surprising experimental finding that myosin walks at a faster speed but for a shorter distance as the ATP concentration increases in the absence of ADP. It also suggests that under physiological condition ([ADP] approximately 12-50 microM), myosin walks with a higher speed and for longer distances when ATP is more abundant.

The Influence of Insertion of a Critical Residue (Arg356) in Structure and Bioluminescence Spectra of Firefly Luciferase

The firefly bioluminescence reaction, which uses luciferin, Mg-ATP, and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and visible light is emitted. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetle luciferases, those from Phrixothrix railroad worm emit either yellow or red bioluminescence colors. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional Arg residue at 353, which is absent in firefly luciferases. We report here the construction and purification of a mutant at residue Arg(356), which is not conserved in beetle luciferases. By insertion of an additional residue (Arg(356)) using site-specific insertion mutagenesis in a green-emitter luciferase (Lampyris turkestanicus) the color of emitted light was changed to red and the optimum temperature of activity was also increased. Insertion of this Arg in an important flexible loop showed changes of the bioluminescence color and the luciferase reaction took place with relatively retention of its basic kinetic properties such as Km and relative activity. Comparison of native and mutant luciferases using homology modeling reveals a significant conformational change of the flexible loop in the red mutant. Movement of flexible loop brought about a new ionic interaction concomitant with a change in polarity of the emitter site, thereby leading to red emission. It is worthwhile to note that the increased optimum temperature and emission of red light might make mutant luciferase a suitable reporter for the study of gene expression and bioluminescence imaging.

Three tandem HRDC domains have synergistic effect on the RecQ functions in Deinococcus radiodurans

The RecQ family of DNA helicases performs essential functions in the maintenance of genomic stability in all organisms. In Deinococcus radiodurans, DR1289 is a special member of RecQ family with unique arrangement of three tandem HRDC domains in the C-terminus. A dr1289 mutant is hypersensitive to γ-irradiation, UV, H 2O 2 and mitomycin C. By complementing the dr1289 mutant with various domains of Dr1289 in vivo, we have determined that the helicase and all three HRDC domains are indispensable for complete DNA damage resistance. Using a continuous fluorescent dye-displacement assay, we investigated the optimal conditions for Dr1289 unwinding function at various concentrations of ATP and metal ions to show that the helicase activity is comparable to what observed in Escherichia coli RecQ. We also found that the helicase domain is necessary for the unwinding and ATPase activity and that the three tandem HRDC domains increase the efficiency of these activities. Based on these data, we propose that the C-terminus of Dr1289 has evolved in D. radiodurans to confront the types and amounts of DNA damage.