ATP Gamma Research Articles

Identification of virulence-associated factors in Vibrio parahaemolyticus with special reference to moonlighting protein: a secretomics study.

Vibrio parahaemolyticus causes seafood-borne gastroenteritis infection in human which can even lead to death. The pathogenic strain of V. parahaemolyticus secretes different types of virulence factors thatare directly injected into the host cell by a different type of secretion system which helps bacteria to establish its own ecological niche within the organism. Therefore, the aim of this study was to isolate the extracellular secreted proteins from the trh positive strain of V. parahaemolyticus and identify them using two-dimensional gel electrophoresis and MALDI-TOFMS/MS. Seventeen different cellular proteins viz, Carbamoyl-phosphate synthase, 5-methyltetrahydropteroyltriglutamate, tRNA-dihydrouridine synthase, Glycerol-3-phosphate dehydrogenase, Orotidine 5'-phosphate decarboxylase, Molybdenum import ATP-binding protein, DnaJ, DNA polymerase IV, Ribosomal RNA small subunit methyltransferase G, ATP synthase subunit delta and gamma, Ribosome-recycling factor, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, tRNA pseudouridine synthase B, Ditrans, polycis-undecaprenyl-diphosphate synthase, Oxygen-dependent coproporphyrinogen-III oxidase, and Peptide deformylase 2 were identified whichare mainly involved in different metabolic and biosynthetic pathways. Furthermore, the molecular function of the identified proteins were associated with catalytic activity, ligase activity, transporter, metal binding, and ATP synthase when they are intercellular. However, to understand the importance of these secreted proteins in the infection and survival of bacteria inside the host cell, pathogen-host protein-protein interactions (PPIs) were carried out which identified the association of eight secreted proteins with 41 human proteins involved in different cellular pathways, including ubiquitination degradation, adhesion, inflammation, immunity, and programmed cell death. The present study provides unreported strategies on host-cell environment's survival and adaptation mechanisms for the successful establishment of infections and intracellular propagation.

Crystal Structures of N-terminal Domain of Human Hsp90 With ATP Analogues Reveal The Functional Regulation of Hsp90*

Heat shock protein 90 (Hsp90) is essential for folding, maturation and stabilization of many important proteins, which are involved in cell cycle regulation, signal transduction, and cell growth regulation. The highly conserved N-terminal domain contains an ATP binding cleft and thus is responsible for the catalytic activity of Hsp90. In order to further study the function and structure of Hsp90, the N-terminal of the human Hsp90 was cocrystallized with AMPPNP and ATP gamma S. The cocrystallization experiments were carried out at 277K using the hanging drop vapor-diffusion method, X-ray diffraction data were collected on beamline 17U at the SSRF and the structures were solved by molecular replacement. The densities of the two nucleotides were captured and the interactions between Hsp90(N) and nucleotides were clearly described. We confirmed that the gamma-phosphate of ATP gamma S was not hydrolyzed by Hsp90(N). The position of S 1 and ATP lid in human Hsp90(N)-AMPPNP differs significantly from that of the structure of yeast Hsp90-AMPPNP. By analyzing the structure of human Hsp90(N)-AMPPNP, we found that the interactions of E18-K100 and N40-D127 block the moving of Si and ATP lid, and then prevent the dimerization of Hsp90(N). This reflects the complexity and coordination of Hsp90 on the regulation of the function.

Individual Actin Filaments in a Microfluidic Flow Reveal the Mechanism of ATP Hydrolysis and Give Insight Into the Properties of Profilin

Author SummaryActin proteins assemble into microfilaments that control cell shape and movement by polymerizing or depolymerizing. These actin monomers can bind ATP or ADP molecules. The incorporation of an ATP-actin monomer into a growing filament results in rapid cleavage of ATP into ADP and inorganic phosphate (Pi), followed by a slower release of Pi. As a consequence, actin filaments are composed mainly of ADP- and ADP-Pi-actin subunits, which have different depolymerization kinetics and mechanical properties, and can be targeted specifically by regulatory proteins that affect filament function. Hence, the understanding of many cellular processes requires a knowledge of the ADP/ADP-Pi composition of actin filaments at a molecular scale. This has so far remained elusive because traditional studies rely on measuring an average over many filaments in solution. To address this issue, we developed a microfluidics setup to monitor individual filaments with light microscopy while rapidly changing their chemical environment. We find that depolymerization accelerates progressively and corresponds to an exponential ADP-Pi-actin profile in the filament, meaning that each subunit releases its Pi with the same rate. Our method also provides novel insight into the function of profilin, a protein important for regulation of actin dynamics in cells, thus demonstrating the method's potential in the functional analysis of actin regulators.

At the Source: Treating Heart Failure by Altering Muscle Motor Function

### Cardiac Myosin Activation: A Potential Therapeutic Approach for Systolic Heart Failure Malik et al Science . 2011;331:1439–1443. A new study in Science provides proof of the principle for directly modulating the cardiac muscle's motor, myosin, as a therapeutic target in heart failure. The human and economic tolls from heart failure (HF) are significant in both developed and undeveloped countries.1–3 Although HF can take many forms,4,5 it is most commonly associated with decreased cardiac contractility and is, therefore, referred to as systolic HF (sHF).6 Current therapeutic strategies rely on blocking neurohormonal activation by inhibiting associated pathways or by receptor blockade using either β-adrenergic or aldosterone blockers.7,8 Alternatively, some therapies are directed at increasing cardiac contractility in an effort to restore sufficient blood flow, usually by activating second messenger signaling pathways that increase cardiomyocyte calcium levels and augment sarcomere contraction.9 Both strategies suffer from a lack of specificity in that they target proteins upstream of the actual contractile events that underlie force production. Affecting important signaling pathways or receptor-ligand interactions can impact cellular function and perturb homeostasis in ways unrelated to the contractile apparatus, leading to undesirable side effects, such as hypotension, tachycardia, and even arrhythmias that can lead to sudden death.10 Recognizing the significant limitations of current therapeutic modalities, Malik and co-workers have directed their efforts at directly modulating cardiac contractility by tuning motor function at the source, the myosin heavy chain. The cardiac myosin heavy chains are encoded by two genes, MYH6 , which encodes α myosin heavy chain, and MYH7 , which encodes β myosin heavy chain, the latter being the predominant isoform in human ventricle. Although having greater than 90% amino acid homology, the two proteins differ significantly at the biochemical level. In comparison with α myosin, β myosin is relatively more efficient in force production as a function of energy consumption, with …

Convergent donor and acceptor substrate utilization among kinase ribozymes

Accommodation of donor and acceptor substrates is critical to the catalysis of (thio)phosphoryl group transfer, but there has been no systematic study of donor nucleotide recognition by kinase ribozymes, and there is relatively little known about the structural requirements for phosphorylating internal 2′OH. To address these questions, new self-phosphorylating ribozymes were selected that utilize ATP(gammaS) or GTP(gammaS) for 2′OH (thio)phosphorylation. Eight independent sequence families were identified among 57 sequenced isolates. Kinetics, donor nucleotide recognition and secondary structures were analyzed for representatives from each family. Each ribozyme was highly specific for its cognate donor. Competition assays with nucleotide analogs showed a remarkable convergence of donor recognition requirements, with critical contributions to recognition provided by the Watson–Crick face of the nucleobase, lesser contributions from donor nucleotide ribose hydroxyls, and little or no contribution from the Hoogsteen face. Importantly, most ribozymes showed evidence of significant interaction with one or more donor phosphates, suggesting that—unlike most aptamers—these ribozymes use phosphate interactions to orient the gamma phosphate within the active site for in-line displacement. All but one of the mapped (thio)phosphorylation sites are on unpaired guanosines within internal bulges. Comparative structural analysis identified three loosely-defined consensus structural motifs for kinase ribozyme active sites.

ATP Dependence of Na+-Driven Cl–HCO3 Exchange in Squid Axons

Squid giant axons recover from acid loads by activating a Na(+)-driven Cl-HCO(3) exchanger. We internally dialyzed axons to an intracellular pH (pH( i )) of 6.7, halted dialysis and monitored the pH(i) recovery (increase) in the presence of ATP or other nucleotides, using cyanide to block oxidative phosphorylation. We computed the equivalent acid-extrusion rate (J(H)) from the rate of pH(i) increase and intracellular buffering power. In experimental series 1, we used dialysis to vary [ATP](i), finding that Michaelis-Menten kinetics describes J (H) vs. [ATP](i), with an apparent V(max) of 15.6 pmole cm(-2 )s(-1) and K (m) of 124 microM. In series 2, we examined ATP gamma S, AMP-PNP, AMP-PCP, AMP-CPP, GMP-PNP, ADP, ADP beta S and GDP beta S to determine if any, by themselves, could support transport. Only ATP gamma S (8 mM) supported acid extrusion; ATP gamma S also supported the HCO (3)(-) -dependent (36)Cl efflux expected of a Na(+)-driven Cl-HCO(3) exchanger. Finally, in series 3, we asked whether any nucleotide could alter J (H) in the presence of a background [ATP](i) of approximately 230 microM (control J (H) = 11.7 pmol cm(-2 )s(-1)). We found J (H) was decreased modestly by 8 mM AMP-PNP (J (H) = 8.0 pmol cm(-2 )s(-1)) but increased modestly by 1 mM ADP beta S (J (H) = 16.0 pmol cm(-2 )s(-1)). We suggest that ATP gamma S leads to stable phosphorylation of the transporter or an essential activator.

Protein Remodeling by ClpB AAA+ ATPase Independent of the DnaK/Hsp70 Chaperone System

AAA+ ATPases forcibly remodel diverse macromolecular structures. ClpB, a member of the AAA+ family of ATPases and a Clp/Hsp100 chaperone, is a hexameric protein containing two AAA+ domains and an axial channel spanning the length of the hexamer. ClpB is required for cell survival after exposure to extreme thermal stress and functions by dissolving and renaturing aggregated proteins. ClpB acts in protein disaggregation in conjunction with the DnaK chaperone and its two co-chaperones, DnaJ and GrpE, both in vivo and in vitro. Remarkably, when given mixtures of ATP and ATP gamma S (a poorly hydrolysable ATP analog), ClpB is activated to perform protein remodeling functions independent of the DnaK chaperone system in vitro. This finding has allowed us to study ClpB activities in isolation, without the complicating effects of DnaK, DnaJ and GrpE. We have compared ClpB to other Clp/Hsp100 proteins, including ClpA and ClpX, in protein activation using the model “small aggregate”, P1 RepA, in protein unfolding using GFP fusion proteins, and in protein renaturation using heat-aggregated proteins. We have found that in some aspects ClpB is like ClpA and ClpX and in others it is somewhat different.

A DEAD Protein that Activates Intron Self-Splicing without Unwinding RNA

The group II intron ai5gamma from S. cerevisiae requires high temperature and salt to self-splice in vitro, but it is assisted by the protein Mss116 in vivo. Here we show that Mss116 can stimulate splicing of ai5gamma under near-physiological conditions in vitro, which represents one of the first cases in which a DExH/D protein is shown to act on its natural target. Importantly, we demonstrate that a small subset of DEAD-box proteins can also stimulate ai5gamma splicing in vitro and may represent a distinct subfamily of DEAD-box proteins that functions in RNA tertiary structure assembly. Mutational analysis shows that while ATPase activity is required for stimulation of splicing by Mss116, helicase activity is not. This finding indicates that Mss116 is unlikely to promote intron splicing through the unwinding of kinetic traps. Rather, we propose that Mss116 promotes the ordered assembly of large RNA molecules through stabilization of on-pathway intermediates.

A single mismatch in the DNA induces enhanced aggregation of MutS

Changes in the oligomeric status of MutS protein was probed in solution by dynamic light scattering (DLS), and corroborated by sedimentation analyses. In the absence of any nucleotide cofactor, free MutS protein [hydrodynamic radius (Rh) of 10-12 nm] shows a small increment in size (Rh 14 nm) following the addition of homoduplex DNA (121 bp), whereas the same increases to about 18-20 nm with heteroduplex DNA containing a mismatch. MutS forms large aggregates (Rh > 500 nm) with ATP, but not in the presence of a poorly hydrolysable analogue of ATP (ATPgammaS). Addition of either homo- or heteroduplex DNA attenuates the same, due to protein recruitment to DNA. However, the same protein/DNA complexes, at high concentration of ATP (10 mm), manifest an interesting property where the presence of a single mismatch provokes a much larger oligomerization of MutS on DNA (Rh > 500 nm in the presence of MutL) as compared to the normal homoduplex (Rh approximately 100-200 nm) and such mismatch induced MutS aggregation is entirely sustained by the ongoing hydrolysis of ATP in the reaction. We speculate that the surprising property of a single mismatch, in nucleating a massive aggregation of MutS encompassing the bound DNA might play an important role in mismatch repair system.

ATP sensitivity of ATP‐sensitive K+ channels: role of the γ phosphate group of ATP and the R50 residue of mouse Kir6.2

ATP-sensitive K (K(ATP)) channels are composed of Kir6, the pore-forming protein, and the sulphonylurea receptor SUR, a regulatory protein. We and others have previously shown that positively charged residues in the C terminus of Kir6.2, including R201 and K185, interact with the alpha and beta phosphate groups of ATP, respectively, to induce channel closure. A positively charged residue in the N terminus, R50, is also important, and has been proposed to interact with either the gamma or beta phosphate group of ATP. To examine this issue, we systematically mutated R50 to residues of different size, charge and hydropathy, and examined the effects on adenine nucleotide sensitivity in the absence and presence of SUR1. In the absence of SUR1, only the size of residue 50 significantly altered ATP sensitivity, with smaller side chains decreasing ATP sensitivity. In the presence of SUR1, however, hydrophathy and charge also played a role. Hydrophilic residues decreased ATP sensitivity more than hydrophobic residues for small size residues, and, surprisingly, negatively charged residues E and D preserved ATP sensitivity and increased ADP sensitivity relative to the wild-type residue R. These observations suggest that a negative charge near position 50, due to either mutation of R50 or the interaction of the gamma phosphate group of ATP with R50, facilitates closure of the ATP-dependent gate. Mutation of the nearby positively charged residue R54, known to be involved in stabilizing channel opening via electrostatic interactions with phosphatidylinositol 4,5-bisphosphate (PIP2), also caused increased ADP sensitivity as compared with ATP, suggesting a loss of function of ATP's gamma phosphate. Based on these results, we propose that a phosphate group or a negative charge at position 50 initiates channel closure by destabilizing the electrostatic interactions between negative phosphate groups of PIP2 and residues such as R54.

Molecular Adaptation in the Ice Worm, Mesenchytraeus solifugus: Divergence of Energetic-Associated Genes

The ice worm, Mesenchytraeus solifugus, is the largest glacially obligate metazoan and among only a few metazoan species that complete their life cycle at 0 degrees C. We conducted a large-scale sequencing analysis of cDNAs isolated from ice worm anterior segments. Sequence comparisons among an available group of ice worm, arthropod, chordate, and nematode homologues suggest that ice worms encode proteins that are less bulky, are less polar, and contain fewer charged residues. Also, subunits of the catalytic F1 ATP synthase complex appear to have diverged more rapidly than other ice worm genes examined, suggesting a role in cold-temperature adaptation. Modeling of F1 ATP synthase beta and gamma subunits identified nonconservative, ice worm-specific amino acid substitutions at subunit contact sites and at sites proximal to the catalytic site.

Characterization of purine receptors in mouse thoracic aorta.

The contracting and relaxing effects of purines and UTP were investigated on rings of mouse thoracic aorta in vitro. UTP, ATP gamma S, and alpha-beta-Methyleneadenosine 5'triphosphate contracted rings with and without endothelium. On the contrary, adenosine, AMP, ADP, ATP, and 2-(methylthio)adenosine 5'-diphosphate had no effect on relaxed rings. When rings were tonically contracted by U46619 a thromboxane A2 analogue, ATP, ADP, ATP gamma S, 2-(methylthio)adenosine 5'-diphosphate, and UTP caused endothelium-dependent but not independent relaxations.I conclude that ATP acts on P2Y2 and P2Y1 receptors on the endothelial cells to cause endothelium-dependent relaxation. In this tissue, the relaxing effect of ATP dominates by endothelium-dependent ways when aorta rings are contracted by a stable thromboxane A2 analog. However receptors mediating contraction in response to purines and pyrimidines are present on smooth muscle cells. Indeed, the stimulation of P2Y receptors by UTP as well as the activation of P2X family receptors by ATP gamma S causes a contraction. The potential contractile effect of ATP seems masked by its hydrolysis by ectonucleotidases.

Extracellular ATP-induced calcium signaling in mIMCD-3 cells requires both P2X and P2Y purinoceptors.

Kidney tubules are targets for the activation of locally released nucleotides through multiple P2 receptor types. Activation of these P2 receptors modulates cellular Ca(2+) signaling and downstream cellular function. The purpose of this study was to determine whether P2 receptors were present in mIMCD-3 cells, a mouse inner medullary collecting duct cell line, and if so, to examine their link with intracellular Ca(2+) homeostasis. To monitor intracellular Ca(2+) concentration ([Ca(2+)](i)), experiments were conducted using the fluorescent dye fura 2. ATP (0.1-100 microM) produced a dose-dependent increase in [Ca(2+)](i) in a physiological Ca(2+)-containing solution, with an EC(50) of 2.5 microM. The P2-receptor antagonist PPADS reduced the effect of ATP on [Ca(2+)](i), and the P1-receptor agonist adenosine caused only a small increase in [Ca(2+)](i). Preincubation of cells with the phospholipase C antagonist U-73122 blocked the ATP-induced increase in [Ca(2+)](i), indicating P2Y receptors were involved in this process. In a Ca(2+)-free bath solution, thapsigargin and ATP induced intracellular Ca(2+) release from an identical pool. Nucleotides caused an increase in [Ca(2+)](i) in the potency order of UTP = ATP > ATP gamma S > ADP > UDP that is best fitted with the P2Y(2) subtype profile. Although the P2Y agonist UTP induced a similar large transient increase in [Ca(2+)](i) as did ATP, a small but sustained increase in [Ca(2+)](i) occurred only in ATP-stimulated cells, suggesting the role of P2X receptors in Ca(2+) influx. The sustained increase in [Ca(2+)](i) could be blocked by either nonselective cation channel blockers Gd(3+) or P2X antagonists PPADS and PPNDS. Furthermore, when either Gd(3+) or PPNDS was applied to the bath solution before ATP application, the ATP-induced increase in [Ca(2+)](i) was significantly reduced. Both RT-PCR and Western blotting corroborated the presence of P2X(1) and P2Y(2) receptors. These studies demonstrate that mIMCD-3 cells have both P2X and P2Y subtype receptors and that the activation of both P2X and P2Y receptors by extracellular ATP appears to be required to regulate intracellular Ca(2+) signaling.

Nucleotide-induced switch in oligomerization of the AAA+ ATPase ClpB.

ClpB is a member of the bacterial protein-disaggregating chaperone machinery and belongs to the AAA(+) superfamily of ATPases associated with various cellular activities. The mechanism of ClpB-assisted reactivation of strongly aggregated proteins is unknown and the oligomeric state of ClpB has been under discussion. Sedimentation equilibrium and sedimentation velocity show that, under physiological ionic strength in the absence of nucleotides, ClpB from Escherichia coli undergoes reversible self-association that involves protein concentration-dependent populations of monomers, heptamers, and intermediate-size oligomers. Under low ionic strength conditions, a heptamer becomes the predominant form of ClpB. In contrast, ATP gamma S, a nonhydrolyzable ATP analog, as well as ADP stabilize hexameric ClpB. Consistently, electron microscopy reveals that ring-type oligomers of ClpB in the absence of nucleotides are larger than those in the presence of ATP gamma S. Thus, the binding of nucleotides without hydrolysis of ATP produces a significant change in the self-association equilibria of ClpB: from reactions supporting formation of a heptamer to those supporting a hexamer. Our results show how ClpB and possibly other related AAA(+) proteins can translate nucleotide binding into a major structural transformation and help explain why previously published electron micrographs of some AAA(+) ATPases detected both six- and sevenfold particle symmetry.

ATP Potentiates Interleukin-1β-induced MMP-9 Expression in Mesangial Cells via Recruitment of the ELAV Protein HuR

Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1 beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATP gamma S but conferred a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATP gamma S potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATP gamma S. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATP gamma S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1 beta-inducibility. By confocal microscopy we demonstrate that ATP gamma S increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.

Catalysis and rotation of F1 motor: cleavage of ATP at the catalytic site occurs in 1 ms before 40 degree substep rotation.

F1, a water-soluble portion of FoF1-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central gamma-subunit rotates in the alpha 3 beta 3 cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 80 degrees substep rotation, interim dwell, and rapid 40 degrees substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATP binding, correspond to these events. To discover which steps, we analyzed rotations of F1 subcomplex (alpha 3 beta 3 gamma) from thermophilic Bacillus PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F1 and hydrolysis of a slowly hydrolyzable substrate ATP gamma S (adenosine 5'-[gamma-thio]triphosphate) by wild-type F1. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATP gamma S hydrolysis by the mutant F1 ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on F1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.

Remodeling of Cardiolipin by Phospholipid Transacylation

Mitochondrial cardiolipin (CL) contains unique fatty acid patterns, but it is not known how the characteristic molecular species of CL are formed. We found a novel reaction that transfers acyl groups from phosphatidylcholine or phosphatidylethanolamine to CL in mitochondria of rat liver and human lymphoblasts. Acyl transfer was stimulated by ADP, ATP, and ATP gamma S, but not by other nucleotides. Coenzyme A stimulated the reaction only in the absence of adenine nucleotides. Free fatty acids were not incorporated into CL under the same incubation condition. The transacylation required addition of exogenous CL or monolyso-CL, whereas dilyso-CL was not a substrate. Transacylase activity was decreased in lymphoblasts from patients with Barth syndrome (tafazzin deletion), and this was accompanied by drastic changes in the molecular composition of CL. In rat liver, where linoleic acid was the most abundant residue of CL, only linoleoyl groups were transferred into CL, but not oleoyl or arachidonoyl groups. We demonstrated complete remodeling of tetraoleoyl-CL to tetralinoleoyl-CL in rat liver mitochondria and identified the intermediates linoleoyl-trioleoyl-CL, dilinoleoyl-dioleoyl-CL, and trilinoleoyl-oleoyl-CL by high-performance liquid chromatography. The data suggest that CL is remodeled by acyl specific phospholipid transacylation and that tafazzin is an acyltransferase involved in this mechanism.

Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye.

Ultrasensitive detection of minute amounts of phosphorylated proteins and peptides is a key requirement for unraveling many of the most important signal transduction pathways in mammalian systems. Protein microarrays are potentially useful tools for sensitive screening of global protein expression and post-translational modifications, such as phosphorylation. However, the analysis of signaling pathways has been hampered by a lack of reagents capable of conveniently detecting the targets of protein kinases. Historically, phosphorylation detection methods have relied upon either radioisotopes ((gamma-(32)P)ATP(gamma-(33)P)ATP labeling) or phosphoamino acid-selective antibodies. Both of these methods suffer from relatively well-known shortcomings. In this study, a small molecule fluorophore phosphosensor technology is described, referred to as Pro-Q Diamond dye, which is capable of ultrasensitive global detection and quantitation of phosphorylated amino acid residues in peptides and proteins displayed on microarrays. The utility of the fluorescent Pro-Q Diamond phosphosensor dye technology is demonstrated using phosphoproteins and phosphopeptides as well as with protein kinase reactions performed in miniaturized microarray assay format. Instead of applying a phosphoamino acid-selective antibody labeled with a fluorescent or enzymatic tag for detection, a small, fluorescent probe is employed as a universal sensor of phosphorylation status. The detection limit for phosphoproteins on a variety of different commercially available protein array substrates was found to be 312-625 fg, depending upon the number of phosphate residues. Characterization of the enzymatic phosphorylation of immobilized peptide targets with Pro-Q Diamond dye readily permits differentiation between specific and non-specific peptide labeling at picogram to subpicogram levels of detection sensitivity.

Biochemical Evidence for Interaction between the Two Nucleotide Binding Domains of ArsA: INSIGHTS FROM MUTANTS AND ATP ANALOGS

ArsA, the peripheral membrane component of the anion-translocating ATPase ArsAB, consists of two nucleotide binding domains (A1 and A2), which are connected by a linker sequence. Previous studies on ArsA have focused on the function of each nucleotide binding domain and the role of the linker, whereas the present study looks at the interactions between the binding domains and their interactions with the linker. It has previously been shown that the A1 domain of ArsA carries out unisite catalysis in the absence of antimonite, while A2 is recruited in multisite catalysis by antimonite in the presence of a functional A1 domain. Multisite catalysis thus seems to result from an interaction between A1 and A2 brought about by antimonite. In the present study, we provide direct biochemical evidence for interaction between the two nucleotide binding domains and show that the linker region acts as a transducer of the conformational changes between them. We find that nucleotide binding to the A2 domain results in a significant, detectable change in the conformation of the A1 domain. Two ATP analogs, FSBA and ATP gamma S, used in this study, were both found to bind preferentially to the A2 domain, and their binding resulted in changing the otherwise compact A1 domain into an open conformation. Point mutations in the A2 domain and the linker region also produced a similar effect on the conformation of A1, thus suggesting that events at A2 are relayed to A1 via the linker. We propose that nucleotide binding to A2 produces a two-tiered conformational change. The significance of these changes in the mechanism of ArsA is discussed.

Kinetic characterization of the weak binding states of myosin V.

Myosin V is a molecular motor shown to move processively along actin filaments. We investigated the properties of the weak binding states of monomeric myosin V containing a single IQ domain (MV 1IQ) to determine if the affinities of these states are increased as compared to conventional myosin. Further, using a combination of non-hydrolyzable nucleotide analogues and mutations that block ATP hydrolysis, we sought to probe the states that are populated during ATP-induced dissociation of actomyosin. MV 1IQ binds actin with a K(d) = 4 microM in the presence of ATP gamma S at 50 mM KCl, which is 10-20-fold tighter than that of nonprocessive class II myosins. Mutations within the switch II region trapped MV 1IQ in two distinct M.ATP states with very different actin binding affinities (K(d) = 0.2 and 2 microM). Actin binding may change the conformation of the switch II region, suggesting that elements of the nucleotide binding pocket will be in a different conformation when bound to actin than is seen in any of the myosin crystal structures to date.