Kinetics Of Nucleotide Binding Research Articles

Influence of expression and purification protocols on Gα biochemical activity: kinetics of plant and mammalian G protein cycles.

Heterotrimeric G proteins are a class of signal transduction complexes with broad roles in human health and agriculturally important plant traits. In the classic paradigm, guanine nucleotide binding to the Gα subunit regulates the activation status of the complex. Using the Arabidopsis thaliana Gα subunit, GPA1, we developed a rapid StrepII-tag mediated purification method that facilitates isolation of protein with increased enzymatic activities as compared to conventional methods, and is demonstrably also applicable to mammalian Gα subunits. We subsequently utilized domain swaps of GPA1 and human GNAO1 to demonstrate the instability of recombinant GPA1 is a function of the interaction between the Ras and helical domains, and can be partially uncoupled from the rapid nucleotide binding kinetics displayed by GPA1.

Examination of the Role of Mg2+ in the Mechanism of Nucleotide Binding to the Monomeric YME1L AAA+ Domain.

The first line of defense in the mitochondrial quality control network involves the stress response from a family of ATP-dependent proteases. We have reported that a solubilized version of the mitochondrial inner membrane ATP-dependent protease YME1L displays nucleotide binding kinetics that are sensitive to the reactive oxygen species hydrogen peroxide under a limiting ATP concentration. Our observations were consistent with an altered YME1L conformational ensemble leading to increased nucleotide binding site accessibility under oxidative stress conditions. To examine this hypothesis further, we report here the results of a comprehensive study of the thermodynamic and kinetic properties underlying the binding of nucleoside di- and triphosphate to the isolated YME1L AAA+ domain (YME1L-AAA+). A combination of fluorescence titrations, molecular dynamics, and stopped-flow fluorescence experiments have demonstrated similarity between nucleotide binding behaviors for YME1L under oxidative conditions and the isolated AAA+ domain. Our data demonstrate that YME1L-AAA+ binds ATP and ADP with affinities equal to ∼30 and 5 μM, respectively, in the absence of Mg2+. We note a negative heterotropic linkage effect between Mg2+ and ATP that arises as the MgCl2 concentration is increased such that the affinity of YME1L-AAA+ for ATP decreases to ∼60 μM in the presence of 10 mM MgCl2. Molecular dynamics methods allow for structural rationalization by revealing condition-dependent conformational populations for YME1L-AAA+. Taken together, these data suggest a preliminary model in which YME1L modulates its affinity for the nucleotide to stabilize against degradation or instability inherent to such stress conditions.

Nucleotide binding kinetics and conformational change analysis of tissue transglutaminase with switchSENSE.

Function, activity, and interactions of proteins crucially depend on their three-dimensional structure and are often regulated by effector binding and environmental changes. Tissue transglutaminase (Transglutaminase 2, TG2) is a multifunctional protein, allosterically regulated by nucleotides and Ca2+ ions, which trigger opposing conformational changes. Here we introduce switchSENSE as a versatile tool for TG2 characterization and provide novel insights into protein conformation as well as analyte binding kinetics. For the first time, we succeeded in measuring the kinetic rate constants and affinities (kon, koff, KD) for guanosine nucleotides (GMP, GDP, GTP, GTPγS). Further, the conformational changes induced by GDP, Ca2+ and the covalent inhibitor Z-DON were observed by changes in TG2's hydrodynamic diameter. We confirmed the well-known compaction by guanosine nucleotides and extension by Ca2+, and provide evidence for TG2 conformations so far not described by structural analysis. Moreover, we analyze the influence of the peptidic Z-DON inhibitor and the R580A mutation on the conformational responsiveness of TG2 to its natural effectors. In summary, this work shows how the combination of structural and kinetic information obtained by switchSENSE opens new perspectives for the characterization of conformationally active proteins and their interactions with ligands, e.g. potential drug candidates.

The Kinetics of Nucleotide Binding to Isolated Chlamydomonas Axonemes Using UV-TIRF Microscopy

Cilia and flagella are long, slender organelles found in many eukaryotic cells, where they have sensory, developmental, and motile functions. All cilia and flagella contain a microtubule-based structure called the axoneme. In motile cilia and flagella, which drive cell locomotion and fluid transport, the axoneme contains, along most of its length, motor proteins from the axonemal dynein family. These motor proteins drive motility by using energy derived from the hydrolysis of ATP to generate a bending wave, which travels down the axoneme. As a first step toward visualizing the ATPase activity of the axonemal dyneins during bending, we have investigated the kinetics of nucleotide binding to axonemes. Using a specially built ultraviolet total internal reflection fluorescence microscope, we found that the fluorescent ATP analog methylanthraniloyl ATP (mantATP), which has been shown to support axonemal motility, binds all along isolated, immobilized axonemes. By studying the recovery of fluorescence after photobleaching, we found that there are three mantATP binding sites: one that bleaches rapidly (time constant ≈ 1.7 s) and recovers slowly (time constant ≈ 44 s), one that bleaches with the same time constant but does not recover, and one that does not bleach. By reducing the dynein content in the axoneme using mutants and salt extraction, we provide evidence that the slow-recovering component, but not the other components, corresponds to axonemal dyneins. The recovery rate of this component, however, is too slow to be consistent with the activation of beating observed at higher mantATP concentrations; this indicates that the dyneins may be inhibited due to their immobilization at the surface. The development of this method is a first step toward direct observation of the traveling wave of dynein activity.

The Effect of Magnesium Ion Concentration on the Nucleotide Specificity and Fidelity of HIV-1 Reverse Transcriptase

Like other DNA polymerases there are two Mg2+ bound to the polymerase site of HIV-1 reverse transcriptase (HIVRT) and they are thought to facilitate the incorporation of normal nucleotides through a classical two metal ion mechanism. However, the effect of Mg2+ on the fidelity of HIVRT and role of each Mg2+ on the steps governing nucleotide incorporation are unknown and although most in vitro measurements have been performed using 6 -10 mM Mg2+, it is reported that the physiological concentration of free Mg2+ may be as low as 0.25 mM. In this study, we investigated the effects of Mg2+ on nucleotide specificity by defining the kinetic parameters governing each step involved in the normal nucleotide in the incorporation pathway; namely, ground-state binding, substrate-induced conformational change, substrate release, chemistry and pyrophosphate release. One Mg2+ is chelated by the nucleotide, which binds to the enzyme as a Mg-dNTP2- complex, while the second Mg2+ binds in a separate step. Surprisingly, Mg2+ concentrations ranging from 0.25 to 10 mM did not affect the kinetics of nucleotide binding or the rate of the conformational change. Rather, the main effect of free Mg2+ is on the rate of the chemistry step. These results demonstrate that the second Mg2+ binds after the conformational change and with an apparent Kd of 4.7 mM. We also measured the fidelity of HIV-1 RT under various concentrations of Mg2+ and show that Mg2+ does not significantly affect the fidelity at concentrations ≥ 0.25 mM. We also examined the effect of Mg2+ concentration on the kinetics of DNA binding. Overall, the results from our study provide valuable information regarding magnesium concentration and nucleotide specificity.

Metal cation controls myosin and actomyosin kinetics

We have perturbed myosin nucleotide binding site with magnesium-, manganese-, or calcium-nucleotide complexes, using metal cation as a probe to examine the pathways of myosin ATPase in the presence of actin. We have used transient time-resolved FRET, myosin intrinsic fluorescence, fluorescence of pyrene labeled actin, combined with the steady state myosin ATPase activity measurements of previously characterized D.discoideum myosin construct A639C:K498C. We found that actin activation of myosin ATPase does not depend on metal cation, regardless of the cation-specific kinetics of nucleotide binding and dissociation. The rate limiting step of myosin ATPase depends on the metal cation. The rate of the recovery stroke and the reverse recovery stroke is directly proportional to the ionic radius of the cation. The rate of nucleotide release from myosin and actomyosin, and ATP binding to actomyosin depends on the cation coordination number.

Insights into the Mechanism of Myosin V Processivity from Point Mutations in the Converter Domain

Dimeric myosin V is an intracellular transporter, which moves cargoes towards the barbed end of actin filaments. Its ability to perform as individual molecules implies that the ATPase cycles in two head domains are coordinated, ensuring the efficient unidirectional motility. Directional loads were shown to modulate the kinetics of nucleotide binding to myosin V, suggesting that the head-head communication may be achieved via intramolecular load, generated when both heads are bound to actin. Here we directly tested the effect of the intramolecular load on the processive stepping of myosin V, using point mutations in the converter domain. The converter is a compact structure, which transmits tiny conformational changes at the nucleotide-binding site to the lever arm. To disturb the transmission mechanism, we replaced with alanines, one at a time, two phenylalanine residues that form a hydrophobic cluster with the C-terminus of the relay helix. These mutations are inferred to reduce intramolecular load but affect neither the nucleotide binding nor actin affinity. We used the combination of bulk kinetic and single-molecule measurements to study in detail the effect of the mutations in the converter on the myosin V performance. The F697A mutation, which completely eliminates intramolecular load, abolishes the motility of myosin V dimers. At the same time, the F749A mutation, which only partly reduces intramolecular load, significantly increases the proportion of backward steps. The obtained results provide strong experimental evidence that the efficient unidirectional processive stepping of myosin V is ensured by the head-head communication based on the intramolecular load.

Fast kinetics of nucleotide binding to Clostridium perfringens family II pyrophosphatase containing CBS and DRTGG domains

We earlier described CBS-pyrophosphatase of Moorella thermoacetica (mtCBS-PPase) as a novel phosphohydrolase that acquired a pair of nucleotide-binding CBS domains during evolution, thus endowing the protein with the capacity to be allosterically regulated by adenine nucleotides (Jämsen, J., Tuominen, H., Salminen, A., Belogurov, G. A., Magretova, N. N., Baykov, A. A., and Lahti, R. (2007) Biochem. J., 408, 327-333). We herein describe a more evolved type of CBS-pyrophosphatase from Clostridium perfringens (cpCBS-PPase) that additionally contains a DRTGG domain between the two CBS domains in the regulatory part. cpCBS-PPase retained the ability of mtCBS-PPase to be inhibited by micromolar concentrations of AMP and ADP and activated by ATP and was additionally activated by diadenosine polyphosphates (AP(n)A) with n > 2. Stopped-flow measurements using a fluorescent nucleotide analog, 2'(3')-O-(N-methylanthranoyl)-AMP, revealed that cpCBS-PPase interconverts through two different conformations with transit times on the millisecond scale upon nucleotide binding. The results suggest that the presence of the DRTGG domain affords greater flexibility to the regulatory part, allowing it to more rapidly undergo conformational changes in response to binding.

Stepping of Myosin V with Point Mutations in the Converter

Myosin V is a dimeric molecular motor, which transports organelles toward the barbed end of actin filaments in cells. Its highly efficient unidirectional motility requires the coordination of ATPase cycles in two head domains, to ensure that the rate-limiting ADP release almost exclusively occurs in the trailing head and the motor steps forward. Single-molecule measurements revealed that the directional loads modulate the kinetics of nucleotide binding to myosin V, suggesting that the head-head communication may be based on intramolecular load, generated when both heads are bound to actin. Here we directly tested the effect of the intramolecular load on the processive stepping of myosin V, using point mutations in the converter domain, which are inferred to reduce intramolecular load but do not affect the nucleotide binding or actin affinity. The converter is a compact structure, which transmits tiny conformational changes, induced at the nucleotide-binding site in the process of ATP hydrolysis, to the lever arm. To disturb the transmission mechanism, we replaced with alanines, one at a time, two phenylalanine residues that form a hydrophobic cluster with the C-terminus of the relay helix. The effects of the mutations on the myosin's V motility were tested by multiple kinetic and single-molecule assays. We found that the F749A mutation significantly increases the proportion of backward steps, whereas the F697A mutation completely abolishes the processive stepping of myosin V. These results provide strong experimental evidence that the efficient unidirectional processive stepping of myosin V is ensured by the head-head communication based on the intramolecular load, which coordinates ATPase cycles in two motor domains.

Transient Kinetics of Nucleotide Binding to the P-glycoprotein Multidrug Transporter

The P-glycoprotein multidrug transporter (Pgp) is a member of the ABC (ATP-binding cassette) superfamily of proteins, and contains two highly conserved cytosolic nucleotide-binding domains (NBDs). These domains couple ATP hydrolysis to the export of a variety of hydrophobic natural products, chemotherapeutic drugs and peptides. Pgp ATPase activity has been extensively characterized, but many details of the catalytic cycle of ATP hydrolysis remain unexplored. The fluorescent nucleotide analogue, TNP-ADP, interacts with the NBDs of Pgp, and the stoichiometry and affinity of binding were previously determined in our laboratory. Transient kinetic methods are commonly used to investigate the mechanism of molecular processes over a time-scale from milliseconds to hundreds of seconds. In this work, we studied the binding of TNP-ADP to Pgp using rapid stopped-flow kinetics. In these experiments, the binding reaction was monitored by following different fluorescent signals, under pseudo-first-order conditions (excess ligand). The time course of TNP-ADP binding displayed five relaxation times (τi's) over a time-span of 300 sec, which correspond to at least five different transitions. All the relaxation times presented a strong temperature-dependence. The time course of the reaction was analyzed by the computational tool Global Kinetic Explorer (KinTek, USA), using several models of sequential isomerizations after/before the fast bimolecular binding reaction. Also, the parameters were analyzed using the matrix projection operator technique for kinetic data (Bujalowski et al., 2000, J. Mol. Biol. 295:831). Both approaches provide, for the first time, information about the rate constants and fluorescent properties of the diverse intermediates formed during binding of TNP-ADP to Pgp. Elucidation of the details of the interaction of nucleotides with Pgp is of prime importance for formulation of a detailed mechanism of action of the transporter. This research is supported by a grant from the Canadian Cancer Society.

Stepping Mechanism of Myosin V: Insights from the Mutations in the Converter

Dimeric molecular motor myosin V transports cargoes towards the barbed end of actin filaments in cells, coordinating ATPase cycles in its two head domains to ensure the efficient unidirectional motility. Directional loads modulate the kinetics of nucleotide binding to myosin V, suggesting that the head-head communication may be achieved via intramolecular load, generated when both heads are bound to actin. Here, using point mutations in the converter domain, we directly tested the effect of the intramolecular load on the processive stepping of myosin V. The converter is a compact structure, which transmits tiny conformational changes, induced at the nucleotide-binding site in the process of ATP hydrolysis, to the lever arm. To disturb the transmission mechanism, we replaced with alanines, one at a time, two phenylalanine residues that form a hydrophobic cluster with the C-terminus of the relay helix. We found that the F749A mutation, which is inferred to reduce intramolecular load but does not affect the nucleotide binding or actin affinity, significantly increases the proportion of backward steps, providing strong experimental evidence that the efficient unidirectional processive stepping of myosin V and, possibly, other dimeric processive motors, is ensured by the head-head communication based on the intramolecular load, which coordinates ATPase cycles in two motor domains.

Transient Dynamics of the Force-Generating Domain in Myosin During the Recovery Stroke

We have used transient time-resolved FRET (TR2-FRET) to detect structural changes within the relay helix in real time during the myosin recovery stroke. The most intriguing problem in myosin's function is the mechanism of signal transduction between the nucleotide binding site and the force-generating domain of myosin during the recovery stroke. Atomic structures of myosin provide clues, but structural measurements during transient kinetics are needed to reveal the details of this mechanism. Kinetics of nucleotide binding is reliably determined from the fluorescence of mant nucleotides or single Trp myosin mutants (W129, W131). Single Trp mutant W501 has provided insight into structural kinetics in the force-generating domain, but the complex fluorescence signals observed during the recovery stroke do not yield clear structural interpretation. To overcome this problem, we have performed FRET measurements that monitor well-defined structural changes in the relay helix, which is proposed to be a key structural element in myosin's force-generating domain. We engineered two double-Cys myosin mutants in the Cys-lite D. discoideum myosin construct (gift from J. Spudich), with one Cys placed at the C-terminus of the relay helix (K498C) and the other at one of two stable positions in the lower 50K domain (D515C or A639C). We labeled these mutants with a donor-acceptor FRET pair (IAEDANS-DABSYL), then used time-resolved FRET (TR-FRET, detects donor lifetime at nanosecond timescale) to detect interprobe distances in equilibrium states trapped by nucleotide analogs, reflecting pre- and post-powerstroke structures of myosin. We then performed these nanosecond time-resolved measurements repetitively every 0.1 ms, in the transient phase after ATP addition using stopped flow. These transient time-resolved FRET (TR2-FRET) signals showed directly that the relay helix changes from straight to bent conformation in the recovery stroke, from the post-powerstroke to the pre-powerstroke state.

Kinetics of nucleotide binding to the β-subunit (AKR6A2) of the voltage-gated potassium (Kv) channel

The β-subunits of the voltage-gated potassium (Kv) channels modulate the kinetics and the gating of Kv channels and assists in channel trafficking and membrane localization. These proteins are members of the AKR6 family. They share a common (α/β) 8 barrel structural fold and avidly bind pyridine nucleotides. Low catalytic activity has been reported for these proteins. Kinetic studies with rat Kvβ2 revealed that the chemical step is largely responsible for the rate-limitation but nucleotide exchange could also contribute to the overall rate. Herein we report our investigations on the kinetics of cofactor exchange using nucleotide-free preparations of Kvβ2. Kinetic traces measuring quenching of Kvβ2 fluorescence by NADP + were consistent with a two-step binding mechanism which includes rapid formation of a loose enzyme:cofactor complex followed by a slow conformational rearrangement to form a tight final complex. Closing of the nucleotide enfolding loop, which in the crystal structure folds over the bound cofactor, provides the structural basis for this rearrangement. The rate of the loop opening required to release the cofactor is similar for NADPH and NADP + (0.9 min −1) and is of the same order of magnitude as the rate of the chemical step estimated previously from kinetic studies with 4-nitrobenzaldehyde (0.3–0.8 min −1, [S.M. Tipparaju, O.A. Barski, S. Srivastava, A. Bhatnagar, Catalytic mechanism and substrate specificity of the beta-subunit of the voltage-gated potassium channel, Biochemistry 47 (2008) 8840–8854]). Binding of NADPH is accompanied by a second conformational change that might be responsible for a 4-fold higher affinity observed with the reduced cofactor and the resulting difficulty in removing bound NADPH from the protein. These data provide evidence that nucleotide exchange occurs on a seconds-to-minutes time scale and set the upper limit for the maximal possible rate of catalysis by Kvβ2. Slow cofactor exchange is consistent with the role of the β-subunit as a metabolic sensor implicated in tonic regulation of potassium currents.

Mechanism, Regulation, and Functional Properties of Dictyostelium Myosin-1B

Myosin-1B is one of three long tailed class-1 myosins containing an ATP-insensitive actin-binding site in the tail region that are produced in Dictyostelium discoideum. Myosin-1B localizes to actin-rich structures at the leading edge of migrating cells where it has been implicated in the formation and retraction of membrane projections, the recycling of plasma membrane components, and intracellular particle transport. Here, we have used a combination of molecular engineering approaches to describe the kinetic and motile properties of the myosin-1B motor and its regulation by TEDS site phosphorylation. Our results show that myosin-1B is a low duty ratio motor and displays the fastest nucleotide binding kinetics of any of the Dictyostelium class-1 myosins studied so far. Different from Dictyostelium myosin-1D and myosin-1E, dephosphorylated myosin-1B is not inactivated but moves actin filaments efficiently, albeit at an up to 8-fold slower velocity in the in vitro motility assay. A further difference is that myosin-1B lacks the ability to switch between rapid movement and bearing tension upon physiological changes of free Mg2+ ions. In this respect, its motor properties appear to be more closely related to Dictyostelium myosin-2 and rabbit skeletal muscle myosin.

Identification and biochemical characterization of Rap2C, a new member of the Rap family of small GTP-binding proteins

The Rap family of small GTP-binding proteins is composed by four different members: Rap1A, Rap1B, Rap2A and Rap2B. In this work we report the identification and characterization of a fifth member of this family of small GTPases. This new protein is highly homologous to Rap2A and Rap2B, binds labeled GTP on nitrocellulose, and is recognized by a specific anti-Rap2 antibody, but not by an anti-Rap1 antibody. The protein has thus been named Rap2C. Binding of GTP to recombinant purified Rap2C was Mg 2+-dependent. However, accurate comparison of the kinetics of nucleotide binding and release revealed that Rap2C bound GTP less efficiently and possessed slower rate of GDP release compared to the highly homologous Rap2B. Moreover, in the presence of Mg 2+, the relative affinity of Rap2C for GTP was only about twofold higher than that for GDP, while, under the same conditions, Rap2B was able to bind GTP with about sevenfold higher affinity than GDP. When expressed in eukaryotic cells, Rap2C localized at the plasma membrane, as dictated by the presence of a CAAX motif at the C-terminus. We found that Rap2C represented the predominant Rap2 protein expressed in circulating mononuclear leukocytes, but was not present in platelets. Importantly, Rap2C was found to be expressed in human megakaryocytes, suggesting that the protein may be down-regulated during platelets generation. This work demonstrates that Rap2C is a new member of the Rap2 subfamily of proteins, able to bind guanine nucleotides with peculiar properties, and differently expressed by various hematopoietic subsets. This new protein may therefore contribute to the still poorly clarified cellular events regulated by this subfamily of GTP-binding proteins.

Kinetics of structural changes in the relay loop and SH3 domain of myosin

The intrinsic fluorescence of smooth muscle myosin signals conformational changes associated with different catalytic states of the ATPase cycle. To elucidate this relationship, we have examined the pre-steady-state kinetics of nucleotide binding, hydrolysis, and product release in motor domain-essential light chain mutants containing a single endogenous tryptophan, either residue 512 in the rigid relay loop or residue 29 adjacent to the SH3 domain. The intrinsic fluorescence of W512 is sensitive to both nucleotide binding and hydrolysis, and appears to report structural changes at the active site, presumably through a direct connection with switch II. The intrinsic fluorescence of W29 is sensitive to nucleotide binding but not hydrolysis, and does not appear to be tightly linked with structural changes occurring at the active site. We propose that the SH3 domain may be sensitive to conformational changes in the lever arm through contacts with the essential light chain.

Equilibrium and Kinetic Analysis of Nucleotide Binding to the DEAD-Box RNA Helicase DbpA

The Escherichia coli DEAD-box protein A (DbpA) is an RNA helicase that utilizes the energy from ATP binding and hydrolysis to facilitate structural rearrangements of rRNA. We have used the fluorescent nucleotide analogues, mantADP and mantATP, to measure the equilibrium binding affinity and kinetic mechanism of nucleotide binding to DbpA in the absence of RNA. Binding generates an enhancement in mant-nucleotide fluorescence and a corresponding reduction in intrinsic DbpA fluorescence, consistent with fluorescence resonance energy transfer (FRET) from DbpA tryptophan(s) to bound nucleotides. Fluorescent modification does not significantly interfere with the affinities and kinetics of nucleotide binding. Different energy transfer efficiencies between DbpA-mantATP and DbpA-mantADP complexes suggest that DbpA adopts nucleotide-dependent conformations. ADP binds (K(d) approximately 50 microM at 22 degrees C) 4-7 times more tightly than ATP (K(d) approximately 400 microM at 22 degrees C). Both nucleotides bind with relatively temperature-independent association rate constants (approximately 1-3 microM(-1) s(-1)) that are much lower than predicted for a diffusion-limited reaction. Differences in the binding affinities are dictated primarily by the dissociation rate constants. ADP binding occurs with a positive change in the heat capacity, presumably reflecting a nucleotide-induced conformational rearrangement of DbpA. At low temperatures (<22 degrees C), the binding free energies are dominated by favorable enthalpic and unfavorable entropic contributions. At physiological temperatures (>22 degrees C), ADP binding occurs with positive entropy changes. We favor a mechanism in which ADP binding increases the conformational flexibility and dynamics of DbpA.

Mechanism of Nucleotide Binding to Actomyosin VI: EVIDENCE FOR ALLOSTERIC HEAD-HEAD COMMUNICATION

We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescence of pyrene-labeled actin filaments. ATP binds single-headed myosin VI following a two-step reaction mechanism with formation of a low affinity collision complex (1/K(1)' = 5.6 mm) followed by isomerization (k(+2)' = 176 s-1) to a state with weak actin affinity. The rates and affinity for ADP binding were measured by kinetic competition with ATP. This approach allows a broader range of ADP concentrations to be examined than with fluorescent nucleotide analogs, permitting the identification and characterization of transiently populated intermediates in the pathway. ADP binding to actomyosin VI, as with ATP binding, occurs via a two-step mechanism. The association rate constant for ADP binding is approximately five times greater than for ATP binding because of a higher affinity in the collision complex (1/K(5b)' = 2.2 mm) and faster isomerization rate constant (k(+5a)' = 366 s(-1)). By equilibrium titration, both heads of a myosin VI dimer bind actin strongly in rigor and with bound ADP. In the presence of ATP, conditions that favor processive stepping, myosin VI does not dwell with both heads strongly bound to actin, indicating that the second head inhibits strong binding of the lead head to actin. With both heads bound strongly, ATP binding is accelerated 2.5-fold, and ADP binding is accelerated >10-fold without affecting the rate of ADP release. We conclude that the heads of myosin VI communicate allosterically and accelerate nucleotide binding, but not dissociation, when both are bound strongly to actin.

Binding of SecA to the SecYEG Complex Accelerates the Rate of Nucleotide Exchange on SecA

SecYEG translocase mediates the transport of preproteins across the inner membrane of Escherichia coli. SecA binds the membrane-embedded SecYEG protein-conducting channel with high affinity and then drives the stepwise translocation of preproteins across the membrane through multiple cycles of ATP binding and hydrolysis. We have investigated the kinetics of nucleotide binding to SecA while associated with the SecYEG complex. Lipid-bound SecA was separated from Se-cYEG-bound SecA by sedimentation of the proteoliposomes through a glycerol cushion, which maintains the SecA native state and effectively removes the lipid-bound SecA fraction. Nucleotide binding was assessed by means of fluorescence resonance energy transfer using fluorescent ATP analogues as acceptors of the intrinsic SecA tryptophan fluorescence in the presence of a tryptophanless variant of the SecYEG complex. Binding of SecA to the SecYEG complex elevated the rate of nucleotide exchange at SecA independently of the presence of preprotein. This defines a novel pretranslocation activated state of SecA that is primed for ATP hydrolysis upon preprotein interaction.

Kinetic mechanism of nucleotide binding toEscherichia coli transcription termination factor Rho: Stopped-flow kinetic studies using ATP and fluorescent ATP analogues

Escherichia coli transcription termination factor Rho catalyzes the unwinding of RNA/DNA duplex in reactions that are coupled to ATP binding and hydrolysis. Fluorescence stopped-flow methods using ATP and the fluorescent 2’(3’)-O-(N-methylanthraniloyl) derivatives (mant-derivatives) of ATP and ADP were used to probe the kinetics of nucleotide binding to and dissociation from the Rho-RNA complex. Presteady state nucleotide binding kinetics provides evidence for the presence of negative cooperativity in nucleotide binding among the multiple nucleotide binding sites on Rho hexamer. The binding of the first nucleotide to the Rho-RNA complex occurs at a bimolecular rate of 3.6×106 M−1sec−1, whereas the second nucleotide binds at a slower rate of 4.7×105 M−1 sec−1 at 18°C. RNA complexed with Rho affects the kinetics of nucleotide interaction with the active sites through conformational changes to the Rho hexamer, allowing the incoming nucleotide to be more accessible to the sites. Adenine nucleotide binding and dissociation is more favorable when RNA is bound to Rho, whereas ATP binding and dissociation step in the absence of RNA occurs significantly slower, at a rate ∼70- and ∼40-fold slower than those observed with the Rho-RNA complex, respectively.