Presence Of Saturating ATP Research Articles

A Cardiomyopathy Mutation in the Myosin Essential Light Chain Alters Actomyosin Structure

We have used site-directed time-resolved fluorescence resonance energy transfer to determine the effect of a pathological mutation in the human ventricular essential light chain (hVELC) of myosin, on the structural dynamics of the actin-myosin complex. The hVELC modulates the function of actomyosin, through the interaction of its N-terminal extension with actin and its C-terminal lobe with the myosin heavy chain. Several mutations in hVELC are associated with hypertrophic cardiomyopathy (HCM). Some biochemical effects of these mutations are known, but further insight is needed about their effects on the structural dynamics of functioning actomyosin. Therefore, we introduced the HCM mutation E56G into a single-cysteine (C16) hVELC construct and substituted it for the VELC of bovine cardiac myosin subfragment 1. Using a donor fluorescent probe on actin (at C374) and an acceptor probe on C16 of hVELC, we performed time-resolved fluorescence resonance energy transfer, directly detecting structural changes within the bound actomyosin complex during function. The E56G mutation has no significant effect on actin-activated ATPase activity or actomyosin affinity in the presence of ATP, or on the structure of the strong-binding S complex in the absence of ATP. However, in the presence of saturating ATP, where both W (prepowerstroke) and S (postpowerstroke) structural states are observed, the mutant increases the mole fraction of the S complex (increasing the duty ratio), while shifting the structure of the remaining W complex toward that of S, indicating a structural redistribution toward the strongly bound (force-generating) complex. We propose that this effect is responsible for the hypercontractile phenotype induced by this HCM mutation in myosin.

Structural Dynamics of Actin during Active Interaction with Myosin depends on the Isoform of the Essential Light Chain

Muscle contraction results from cyclic interaction of myosin and actin, driven by ATP hydrolysis, where the actomyosin complex undergoes transitions between weakly and strongly bound structural states. Rabbit skeletal muscle myosin has two isoforms of the essential light chain (ELC), designated A1 and A2. These isoforms have significant effects on myosin's interaction with actin, resulting in higher catalytic efficiency for A1 but higher in vitro motility and muscle shortening velocity for A2. In order to understand the structural basis of these differences, we have used time-resolved phosphorescence anisotropy (TPA) to investigate the effects of ELC isoforms on the microsecond dynamics of actin during interaction with skeletal muscle myosin in the absence and presence of saturating ATP. Actin was labeled with a phosphorescent probe at C374, and the myosin head (S1) was separated into isoenzymes containing A1 (S1A1) and A2 (S1A2) by ion-exchange chromatography. As previously reported, S1A1 exhibited substantially lower ATPase activity at saturating actin but substantially higher apparent actin affinity, resulting in higher catalytic efficiency. In the absence of ATP, each isoenzyme increased actin's final anisotropy similarly, indicating similar restriction of the amplitude of intrafilament rotational motions in the strong-binding (rigor) state of actomyosin. In contrast, in the presence of saturating ATP, the final anisotropy of actin was substantially higher during interaction with S1A1 than with S1A2. Thus, S1A1 was more effective in restricting actin dynamics during the active interaction of actin and myosin, suggesting greater population of the strong-binding state of actomyosin. We conclude that ELC of skeletal muscle myosin modulates strong-to-weak structural transitions during the actomyosin ATPase cycle in an isoform-dependent manner, and thus modulates the contractile function of actomyosin.

Structural Transitions within the Actomyosin Complex in the Presence of Saturating ATP, Detected by Time-Resolved FRET

Myosin generates force by cycling between actin-attached (strong binding) and actin-detached (weak binding) states during the actomyosin ATPase cycle. However, it remains unclear what specific conformational changes in myosin take place upon binding to actin, and how these structural changes lead to product release and force. To characterize these changes, we have used site-directed FRET to determine distance between specific sites in actin and myosin subfragment 1 (S1) in the presence and absence of ATP. Rabbit skeletal muscle myosin has two essential light chain (ELC) isoforms, A1 and A2.The ELC isoform has significant effects on the interaction of S1 with actin, resulting in higher catalytic efficiency for the S1A1-isoenzyme but higher in vitro motility and muscle shortening velocity for S1A2.We have focused on S1A1, since it binds to actin in the presence of ATP with higher affinity. Actin was labeled at C374 with a donor, AlexaFluor 568. A1 light chain, was expressed,purified and labeled at C177 with an acceptor, AlexaFluor 647. The labeled A1 was exchanged into myosin S1, and S1A1 was isolated using a Talon affinity resin. The efficiency of energy transfer between C374 of actin and C177 of A1 was measured by time-resolved fluorescence, allowing us to detect directly the FRET within the actin-S1A1 complex, even in the presence of saturating ATP. Addition of ATP to the strongly bound complex resulted in a significant decrease in FRET within the weakly bound complex, corresponding to a significant increase in the donor-acceptor distance. We conclude that the transition between strongly and weakly bound states of acto-S1 is associated with a structural transition in the acto-S1 complex that affects the distance from actin to the light-chain domain of myosin.

Direct Observation of the Forward Stepping Motion of Kinesin-1 using Dark-Field Microscopy with 50-Micro Second Temporal Resolution

Kinesin-1 moves along microtubule by alternately moving two motor domains, in which the trailing head detaches and displaces toward the 16-nm forward tubulin-binding site upon ATP binding to the partner head. However, the detailed process of the forward stepping motion is still unclear, mainly due to the lack of temporal resolution of the single-molecule measurements. Here we employed dark-field microscopy with perforated mirror, and observed the movement of one of kinesin heads labeled with 40-nm gold particle with ∼2 nm spatial and ∼55 μs temporal resolutions. In the presence of saturating ATP, the gold particles showed ∼16 nm discrete steps toward the microtubule long axis, which is consistent with the previous observations. However, our measurements also revealed that, just before 16 nm step, the gold particle displaced rightward, perpendicular to the microtubule axis, accompanied with a significant increase in the fluctuation of the gold particle. The rightward asymmetry is consistent with the geometry that the proximal end of the neck linker is located right side of the head. Then the gold particles showed rapid left-forward displacement followed by decrease in the fluctuation, although we could not detect clear sub-step at this temporal resolution. The duration of the highly fluctuating period increased as the ATP concentration was decreased, indicating that the period represents ATP-waiting state. These results provide direct clue to understand how kinesin takes a step forward.

Strain-Dependent Regulation of the Kinesin-1's Catalytic Activity as Studied by Disulfide-Crosslinking of the Neck Linker

Kinesin is a dimeric motor protein that hydrolyzes ATP and moves along microtubules in a hand-over-hand manner. To walk by alternately moving two motor heads, the trailing head should detach from the microtubule prior to the leading head and the detached head should preferentially bind to the forward tubulin-binding site. To explain these mechanisms, we hypothesized that ATP hydrolysis reaction of kinesin motor domain can be regulated depending on the direction of the tension posed to the neck linker: backward strain posed to the neck linker suppresses ATP hydrolysis in the leading head and the forward strain posed to the neck linker suppresses ADP release at the trailing position. To test this hypothesis, we constrained the neck linker in the forward or backward extended conformation using disulfide-crosslinking between cysteine residues on the head and the neck linker, and examined these effects on the microtubule affinity and ADP release kinetics. Single molecule fluorescent observation of the GFP-fused monomeric kinesin showed that when the neck linker was constrained in a backward extended conformation, the dwell time on the microtubule in the presence of saturating ATP was increased by a factor of 15 compared to unconstrained condition. In contrast, stopped-flow measurement showed that when the neck linker was constrained in a forward extended conformation, ADP release rate after microtubule-binding was significantly decreased. These results support the idea that ATP hydrolysis cycle of kinesin's motor domain can be differently regulated depending on the direction of the neck linker extension.

Specific Domains in Yeast Translation Initiation Factor eIF4G Strongly Bias RNA Unwinding Activity of the eIF4F Complex toward Duplexes with 5′-Overhangs

During eukaryotic translation initiation, the 43 S ribosomal pre-initiation complex is recruited to the 5'-end of an mRNA through its interaction with the 7-methylguanosine cap, and it subsequently scans along the mRNA to locate the start codon. Both mRNA recruitment and scanning require the removal of secondary structure within the mRNA. Eukaryotic translation initiation factor 4A is an essential component of the translational machinery thought to participate in the clearing of secondary structural elements in the 5'-untranslated regions of mRNAs. eIF4A is part of the 5'-7-methylguanosine cap-binding complex, eIF4F, along with eIF4E, the cap-binding protein, and the scaffolding protein eIF4G. Here, we show that Saccharomyces cerevisiae eIF4F has a strong preference for unwinding an RNA duplex with a single-stranded 5'-overhang versus the same duplex with a 3'-overhang or without an overhang. In contrast, eIF4A on its own has little RNA substrate specificity. Using a series of deletion constructs of eIF4G, we demonstrate that its three previously elucidated RNA binding domains work together to provide eIF4F with its 5'-end specificity, both by promoting unwinding of substrates with 5'-overhangs and inhibiting unwinding of substrates with 3'-overhangs. Our data suggest that the RNA binding domains of eIF4G provide the S. cerevisiae eIF4F complex with a second mechanism, in addition to the eIF4E-cap interaction, for directing the binding of pre-initiation complexes to the 5'-ends of mRNAs and for biasing scanning in the 5' to 3' direction.

The Neck Linker of Kinesin-1 functions as a Regulator of ATP Hydrolysis Reaction

Kinesin-1 is a highly processive motor that moves along microtubule in a hand-over-hand manner. The neck linker that connects two motor domains has been thought to act as a mechanical amplifier that propels the tethered head forward, however, we recently showed that the neck linker docking is not essential for the forward stepping (Isojima et al. this meeting). We hypothesized that the neck linker docking rather functions to activate ATP hydrolysis reaction. To test this hypothesis, we engineered various monomeric neck linker mutant kinesin and investigated the effect on the ATP hydrolysis reaction. As the neck linker was deleted further from the C-terminus, microtubule-activated ATPase rate was decreased and became almost undetectable when the whole neck linker was removed. The hydrophobic and complementary shaped side-chain of Ile325 is critical for promoting ATP hydrolysis reaction because substitution of this residue alone into glycine caused dramatic decrease in the ATPase rate. Single molecule fluorescent imaging showed that the Ile325G mutant monomer stably bound to the microtubule even in the presence of saturating ATP. A high-resolution cryo-electron microscopic study of the Ile325G mutant in complex with microtubule showed that the kinesin core is rotated clockwise direction even in the presence of saturating AMPPNP, which is similar to the conformation of wild type in the no-nucleotide state. These results suggest that Ile325 is essential for promoting ATP hydrolysis reaction by stabilizing counter clockwise rotation of kinesin motor domain upon ATP binding. This mechanism can also explain the front head gating mechanism for dimeric kinesin: the neck linker of the leading head is pulled backward so that the head cannot proceed ATP hydrolysis cycle until the trailing head detaches from microtubule.

Processive Motility of Heterodimeric Kinesin That Has Defect in the Neck Linker Docking

Kinesin-1 is a motor protein that moves processively along microtubules in a hand-over-hand manner. The neck linker, a short stretch that connects two motor heads, has been shown to undergo ATP-dependent conformational changes, although its role on the processive motility is still controversial. To address this question, we employed a kinesin mutant at switch I (R203K) that can normally bind ATP but is unable to hydrolyse ATP (Klumpp et al. JBC 2003). First we observed the neck linker structural state of monomeric R203K bound to the microtubule using single molecule FRET. We found that the neck linker remained undocked conformation even in the presence of saturating ATP, suggesting that ATP-binding is not sufficient to stabilize neck linker docked state. Next we constructed heterodimeric kinesin that is composed of a wild-type head and a mutant R203K head. As recently been shown by Thoresen and Gelles (Biochemistry 2008), this heterodimer showed slow processive movement along microtubules. Then we observed the conformational changes of this heterodimer using a single molecule FRET sensor as previously developed to distinguish one-head-bound and two-head-bound states (Mori et al. Nature 2007), and found that the heterodimer showed hand-over-hand movement. Unexpectedly, they spent most of the time in the two-head-bound state where wild-type head is in the front and the mutant head is in the rear, indicating that the displacement of rear wild-type head to the forward binding site is not the rate-limiting. These results suggest that the neck linker docking in the microtubule-bound head is not essential for the tethered head to translate and bind to the forward tubulin-binding site and rather is required for promoting ATP-hydrolysis and subsequent detachment of the trailing head from microtubule.

How kinesin waits between steps

Kinesin-1 (conventional kinesin) is a dimeric motor protein that carries cellular cargoes along microtubules by hydrolysing ATP and moving processively in 8-nm steps. The mechanism of processive motility involves the hand-over-hand motion of the two motor domains ('heads'), a process driven by a conformational change in the neck-linker domain of kinesin. However, the 'waiting conformation' of kinesin between steps remains controversial-some models propose that kinesin adopts a one-head-bound intermediate, whereas others suggest that both the kinesin heads are bound to adjacent tubulin subunits. Addressing this question has proved challenging, in part because of a lack of tools to measure structural states of the kinesin dimer as it moves along a microtubule. Here we develop two different single-molecule fluorescence resonance energy transfer (smFRET) sensors to detect whether kinesin is bound to its microtubule track by one or two heads. Our FRET results indicate that, while moving in the presence of saturating ATP, kinesin spends most of its time bound to the microtubule with both heads. However, when nucleotide binding becomes rate-limiting at low ATP concentrations, kinesin waits for ATP in a one-head-bound state and makes brief transitions to a two-head-bound intermediate as it walks along the microtubule. On the basis of these results, we suggest a model for how transitions in the ATPase cycle position the two kinesin heads and drive their hand-over-hand motion.

Temperature-Dependence of Isometric Tension and Cross-Bridge Kinetics of Cardiac Muscle Fibers Reconstituted with a Tropomyosin Internal Deletion Mutant

The effect of temperature on isometric tension and cross-bridge kinetics was studied with a tropomyosin (Tm) internal deletion mutant AS-Δ23Tm (Ala-Ser-Tm Δ(47–123)) in bovine cardiac muscle fibers by using the thin filament extraction and reconstitution technique. The results are compared with those from actin reconstituted alone, cardiac muscle-derived control acetyl-Tm, and recombinant control AS-Tm. In all four reconstituted muscle groups, isometric tension and stiffness increased linearly with temperature in the range 5–40°C for fibers activated in the presence of saturating ATP and Ca 2+. The slopes of the temperature-tension plots of the two controls were very similar, whereas the slope derived from fibers with actin alone had ∼40% the control value, and the slope from mutant Tm had ∼36% the control value. Sinusoidal analysis was performed to study the temperature dependence of cross-bridge kinetics. All three exponential processes A, B, and C were identified in the high temperature range (30–40°C); only processes B and C were identified in the mid-temperature range (15–25°C), and only process C was identified in the low temperature range (5–10°C). At a given temperature, similar apparent rate constants (2 πa, 2 πb, 2 πc) were observed in all four muscle groups, whereas their magnitudes were markedly less in the order of AS-Δ23Tm < Actin < AS-Tm ≈ Acetyl-Tm groups. Our observations are consistent with the hypothesis that Tm enhances hydrophobic and stereospecific interactions (positive allosteric effect) between actin and myosin, but Δ23Tm decreases these interactions (negative allosteric effect). Our observations further indicate that tension/cross-bridge is increased by Tm, but is diminished by Δ23Tm. We conclude that Tm affects the conformation of actin so as to increase the area of hydrophobic interaction between actin and myosin molecules.

Transient kinetics of substrate binding to [formula omitted] measured by fluorescence quenching

This paper examines the transient kinetics of substrate binding to the Na + K + -ATPase labelled with iodoacetamidofluorescein (IAF) using fluorescence quenching by trinitrophenyl-ATP (TNP-ATP). Earlier work (E.H. Hellen, P.R. Pratap, 1996, Fluorescence quenching of IAF- Na + K + -ATPase via energy transfer to TNP-labelled nucleotide, Proceedings of the VIIIth International Conference on the Na + K + -ATPase , in press) has shown that TNP-nucleotide binds to specific sites (from which unlabelled nucleotide can displace it) and nonspecific sites (from which unlabelled nucleotide cannot displace it). Under stopped-flow conditions, quenching of IAF-enzyme fluorescence was well described by a stretched exponential ( F( t) = F ∞ + ΔF exp[− Bt α ]). Physically, this function may be interpreted in terms of its inverse Laplace transform Φ( k). which describes a distribution of rate-constants; α reflects the width of this distribution. As TNP-ATP concentration increased, α decreased, reflecting TNP-ATP binding to sites with higher energy barriers. α decreased by about the same amount with increasing [TNP-ATP] in the presence of saturating ATP, indicating that the distribution of rate-constants is largely associated with the nonspecific binding sites. However, α was significantly less than 1 for ATP-induced fluorescence recovery in the presence of TNP-ATP, indicating that rate-constants associated with specific binding site are also distributed. The distribution of rate-constants for binding to the specific site indicates a distribution in the energy of the transition state for substrate binding. These results suggest that the specific binding site (in either the empty or the full state) may exist in a series of conformations separated by small energy barriers. However, the energy barriers for binding associated with these conformations are significantly distributed.

Purification of the Escherichia coli regulatory protein TyrR and analysis of its interactions with ATP, tyrosine, phenylalanine, and tryptophan.

A plasmid that directs the overexpression of the Escherichia coli regulatory protein TyrR was constructed. Cell extracts of an E. coli strain harboring the plasmid were used to develop a two-step procedure for purifying homogeneous TyrR. The weight-average molecular weight of the pure protein was determined by sedimentation equilibrium analyses to be 110,000 +/- 5,000, indicating that native TyrR is a homodimer. The binding of ligands to TyrR was investigated by the techniques of sedimentation velocity meniscus depletion and steady state dialysis. One mol of ATP bound per mol of TyrR subunit with half-maximal saturation at 5-7 microM ATP. ATP binding curves exhibited positive cooperativity, with a value of 1.3 for the Hill constant, nH. The binding was not significantly affected by the presence of either 500 microM tyrosine or 2 mM phenylalanine. Binding of tyrosine to TyrR (40 microM subunit) could not be detected in the absence of ATP, indicating that the TyrR-tyrosine complex has a dissociation constant (Kd) in excess of 180 microM. However, binding was observed in the presence of saturating ATP (200 microM), where 1 mol of tyrosine bound per mol of TyrR subunit with half-maximal saturation at 50 microM tyrosine. The binding exhibited positive cooperativity (nH of 1.2). There was no detectable binding of either phenylalanine or tryptophan to TyrR (40 microM) in the absence or presence of 200 microM ATP, indicating that any binding of these amino acids to TyrR or TyrR.ATP also has a Kd in excess of 180 microM. Each of these amino acids was found to inhibit the binding of tyrosine by TyrR.ATP when present in large molar excess (20 microM tyrosine and 2 or 10 mM phenylalanine or tryptophan), indicating that TyrR binds each of these amino acids, albeit more weakly than it binds tyrosine.

Controlled proteolysis of ammonia-dependent carbamoyl-phosphate synthetase I from syrian hamster liver

Ammonia-dependent carbamoyl-phosphate synthetase I (carbon-dioxide:ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16; formerly EC 2.7.2.5) isolated from hamster liver mitochondria is comprised of identical 160 kDa polypeptide chains. Controlled proteolysis by elastase sequentially cleaved this molecule into a small number of specific fragments. The first cleavage led to a complete loss of enzymatic activity and the formation of a 145 kDa species that was subsequently degraded into 83 kDa and 62 kDa fragments. Very different results were obtained when proteolysis was carried out in the presence of saturating ATP, MgCl 2, NH 4Cl, and the activator N- acetyl- l-glutamate . These ligands stabilized the molecule 8-fold against elastase digestion. Moreover, only small amounts of the 145 kDa species were generated. Instead, the molecule was initially cleaved into a fully active 120 kDa species anda 40 kDa proteolytic fragment. The same species were found in limit digests conducted in the presence and absence of ligands, indicating that only the sequence of elastase cleavages differed. Comparison of digests conducted in the presence of each ligand alone and in combination, showed that while NH 4Cl and N- acetyl- l-glutamate were necessary for maximal stabilization of the molecule, the altered digestion pattern was produced specifically by MgATP. The MgATP-induced change in digestion pattern correlated well with the steady-state ATP saturation curve, suggesting that the production of the 120 kDa species resulted from ATP binding to the active site. The effect of MgATP on the proteolysis of carbamoyl-phosphate synthetase I was not the result of an alteration in oligomeric structure, but the protection of two elastase cleavage sites. The results were interpreted on the basis of the primary structure recently determined elsewhere.

Interaction between the mitochondrial ATP synthetase and ATPase inhibitor protein: Active/inactive slow pH-dependent transitions of the inhibitor protein

The rate of mitochondrial ATPase inactivation by the naturally occurring inhibitor protein in the presence of saturating ATP and Mg 2+ at pH 8.0 depends hyperbolically on the amount of inhibitor added; the upper limit of an apparent first-order constant for the inactivation process is 1.0 −1 at 25°C. A dramatic difference in the inactivation rate is observed when the protein inhibitor is added to the same assay system from either acidic (pH 4.8) or alkaline (pH 8.2) solutions. The slow reversible transition of the inhibitor from its rapidly reacting ‘acidic’ form to the slow reacting ‘alkaline’ form occurs when the solution of the protein inhibitor is subjected to a pH-jump from 4.8 to 8.2 ( t 1 2 ~ 30 s at 25°C). The pH-profile of the inhibitor active/inactive equilibrium suggests that a group with p K a ~ 6.5 is involved in the transition. Treatment of the inhibitor protein with a histidine-specific reagent (e.g. diethyl pyrocarbonate) abolishes its inactivating effect on the ATPase activity. It is concluded that the protonation/deprotonation of the inhibitor protein followed by its slow conformational changes is the rate-limiting step in the inhibitor-ATP synthetase interaction.

Structure and Function of the Hemoglobins of the Carp, Cyprinus carpio

Abstract Amino acid analysis and tryptic peptide mapping indicate that the α chains of the three major hemoglobin components are significantly different, whereas either no or very slight differences were found among the β chains. The amino acid sequence of the first 28 residues of the β chain of Component I indicates that the A helix is probably the same length in both carp and human β chains. The oxygen equilibria of the isolated components, I, II and III, appear to be indistinguishable from one another. ATP enhances the normal alkaline Bohr effect of carp hemoglobin, and CO2 depresses the Bohr effect between pH 7 and 8. Cooperativity as measured by n in Hill's equation is maximal at about pH 6.5 in 0.05 m bis-(2-hydroxyethyl)-imino-tris-(hydroxymethyl)methane, 0.1 m NaCl but shifts to pH 7.5 in the presence of saturating ATP. A simple model suggests that cooperativity in carp hemoglobin may require the presence of a particular set of protonated species, the concentration of which is maximal at about pH 6.5. The model suggests that the alkaline shift in the position of this maximum in the presence of ATP may result from the preferential binding of ATP at low pH.