Biomedical subjects

Miklós Nyitrai

Publications and source records attributed to Miklós Nyitrai.

14 recordsLinked to original sources

PubMed2006

Conformational changes in actin filaments induced by formin binding to the barbed end.

Formins bind actin filaments and play an essential role in the regulation of the actin cytoskeleton. In this work we describe details of the formin-induced conformational changes in actin filaments by fluorescence-lifetime and anisotropy-decay experiments. The results show that the binding of the formin homology 2 domain of a mammalian formin (mouse mDia1) to actin filaments resulted in a less rigid protein structure in the microenvironment of the Cys374 of actin, weakening of the interactions between neighboring actin protomers, and greater overall flexibility of the actin filaments. The formin effect is smaller at greater ionic strength. The results show that formin binding to the barbed end of actin filaments is responsible for the increase of flexibility of actin filaments. One formin dimer can affect the dynamic properties of an entire filament. Analyses of the results obtained at various formin/actin concentration ratios indicate that at least 160 actin protomers are affected by the binding of a single formin dimer to the barbed end of a filament.

Actin Cytoskeleton↗

PubMed2006

Formins regulate actin filament flexibility through long range allosteric interactions.

The members of the formin family nucleate actin polymerization and play essential roles in the regulation of the actin cytoskeleton during a wide range of cellular and developmental processes. In the present work, we describe the effects of mDia1-FH2 on the conformation of actin filaments by using a temperature-dependent fluorescence resonance energy transfer method. Our results revealed that actin filaments were more flexible in the presence than in the absence of formin. The effect strongly depends on the mDia1-FH2 concentration in a way that indicates that more than one mechanism is responsible for the formin effect. In accordance with the more flexible filament structure, the thermal stability of actin decreased and the rate of phosphate dissociation from actin filaments increased in the presence of formin. The interpretation of the results supports a model in which formin binding to barbed ends makes filaments more flexible through long range allosteric interactions, whereas binding of formin to the sides of the filaments stabilizes the protomer-protomer interactions. These results suggest that formins can regulate the conformation of actin filaments and may thus also modulate the affinity of actin-binding proteins to filaments nucleated/capped by formins.

Actin Cytoskeleton↗

PubMed2005

What limits the velocity of fast-skeletal muscle contraction in mammals?

In rat skeletal muscle the unloaded shortening velocity (Vo) is defined by the myosin isoform expressed in the muscle fibre. In 2001 we suggested that ADP release from actomyosin in solution (controlled by k(-AD)) was of the right size to limit Vo. However, to compare mechanical and solution kinetic data required a series of corrections to compensate for the differences in experimental conditions (0.5 M KCl, 22 degrees C for kinetic assays of myosin, 200 mM ionic strength, 12 degrees C to measure Vo). Here, a method was developed to prepare heavy meromyosin (HMM) from pure myosin isoforms isolated from single muscle fibres and to study k(-AD) (determined from the affinity of the acto-myosin complex for ADP, KAD) and the rate of ATP-induced acto-HMM dissociation (controlled by K1k+2) under the same experimental condition used to measure Vo). In fast-muscle myosin isolated from a wide range of mammalian muscles, k(-AD) was found to be too fast to limit Vo, whereas K1k+2 was of the right magnitude for ATP-induced dissociation of the cross-bridge to limit shortening velocity. The result was unexpected and prompted further experiments using the stopped-flow approach on myosin subfragment-1 (S1) and HMM obtained from bulk preparations of rabbit and rat muscle. These confirmed that the rate of cross-bridge dissociation by ATP limits the velocity of contraction for fast myosin II isoforms at 12 degrees C, while k(-AD) limits the velocity of slow myosin II isoforms. Extrapolating our data to 37 degrees C suggests that at physiological temperature the rate of ADP dissociation may limit Vo for both isoforms.

Adenosine Diphosphate↗

PubMed2005

Kinetic mechanism of myosin IXB and the contributions of two class IX-specific regions.

Myosin IXb (Myo9b) was reported to be a single-headed, processive myosin. In its head domain it contains an N-terminal extension and a large loop 2 insertion that are specific for class IX myosins. We characterized the kinetic properties of purified, recombinant rat Myo9b, and we compared them with those of Myo9b mutants that had either the N-terminal extension or the loop 2 insertion deleted. Unlike other processive myosins, Myo9b exhibited a low affinity for ADP, and ADP release was not rate-limiting in the ATPase cycle. Myo9b is the first myosin for which ATP hydrolysis or an isomerization step after ATP binding is rate-limiting. Myo9b-ATP appeared to be in a conformation with a weak affinity for actin as determined by pyrene-actin fluorescence. However, in actin cosedimentation experiments, a subpopulation of Myo9b-ATP bound F-actin with a remarkably high affinity. Deletion of the N-terminal extension reduced actin affinity and increased the rate of nucleotide binding. Deletion of the loop 2 insertion reduced the actin affinity and altered the communication between actin and nucleotide-binding sites.

Actins↗

PubMed2005

A simple model for the cooperative stabilisation of actin filaments by phalloidin and jasplakinolide.

The stabilisation of magnesium actin filaments by phalloidin and jasplakinolide was studied using the method of differential scanning calorimetry. The results showed that actin could adapt three conformations in the presence of drugs. One conformation was adapted in direct interaction with the drug, while another conformation was identical to that observed in the absence of drugs. A third conformation was induced through allosteric inter-protomer interactions. The effect of both drugs propagated cooperatively along the actin filaments. The number of the cooperative units determined by using a quantitative model was larger for jasplakinolide (15 actin protomers) than for phalloidin (7 protomers).

Actin Cytoskeleton↗

PubMed2004

Adenosine diphosphate and strain sensitivity in myosin motors.

The release of adenosine diphosphate (ADP) from the actomyosin cross-bridge plays an important role in the adenosine-triphosphate-driven cross-bridge cycle. In fast contracting muscle fibres, the rate at which ADP is released from the cross-bridge correlates with the maximum shortening velocity of the muscle fibre, and in some models the rate of ADP release defines the maximum shortening velocity. In addition, it has long been thought that the rate of ADP release could be sensitive to the load on the cross-bridge and thereby provide a molecular explanation of the Fenn effect. However, direct evidence of a strain-sensitive ADP-release mechanism has been hard to come by for fast muscle myosins. The recently published evidence for a strain-sensing mechanism involving ADP release for slower muscle myosins, and in particular non-muscle myosins, is more compelling and can provide the mechanism of processivity for motors such as myosin V. It is therefore timely to examine the evidence for this strain-sensing mechanism. The evidence presented here will argue that a strain-sensitive mechanism of ADP release is universal for all myosins but the basic mechanism has evolved in different ways for different types of myosin. Furthermore, this strain-sensing mechanism provides a way of coordinating the action of multiple myosin motor domains in a single myosin molecule, or in complex assemblies of myosins over long distances without invoking a classic direct allosteric or cooperative communication between motors.

Actins↗

PubMed2004

The effect of phalloidin and jasplakinolide on the flexibility and thermal stability of actin filaments.

In this work the effect of phalloidin and jasplakinolide on the dynamic properties and thermal stability of actin filaments was studied. Temperature dependent fluorescence resonance energy transfer measurements showed that filaments of Ca-actin became more rigid in the presence of phalloidin or jasplakinolide. Differential scanning calorimetric data implied that the stiffer filaments also had greater thermal stability in the presence of phalloidin or jasplakinolide. The fluorescence and calorimetric measurements provided evidences that the extent of stabilization by jasplakinolide was greater than that by phalloidin.

Actins↗

PubMed2004

The core FH2 domain of diaphanous-related formins is an elongated actin binding protein that inhibits polymerization.

Diaphanous-related formins (Drf) are activated by Rho GTP binding proteins and induce polymerization of unbranched actin filaments. They contain three formin homology domains. Evidence as to the effect of formins on actin polymerization were obtained using FH2/FH1 constructs of various length from different Drfs. Here we define the core FH2 domain as a proteolytically stable domain of approximately 338 residues. The monomeric FH2 domains from mDia1 and mDia3 inhibit polymerization of actin and can bind in a 1:1 complex with F-actin at micromolar concentrations. The X-ray structure analysis of the domain shows an elongated, crescent-shaped molecule consisting of three helical subdomains. The most highly conserved regions of the domain span a distance of 75 A and are both required for barbed-end inhibition. A construct containing an additional 72 residue linker has dramatically different properties: It oligomerizes and induces actin polymerization at subnanomolar concentration.

Actins↗

PubMed2003

What kinesin does at roadblocks: the coordination mechanism for molecular walking.

Competing models for the coordination of processive stepping in kinesin can be tested by introducing a roadblock to prevent lead head attachment. We used T93N, an irreversibly binding mutant monomer, as a roadblock, and measured the rates of nucleotide-induced detachment of kinesin monomers or dimers with and without the T93N roadblock using microflash photolysis combined with stopped flow. Control nucleotide-induced monomer (rK340) unbinding was 73.6 s(-1) for ATP and 40.5 s(-1) for ADP. Control ADP-induced dimer (rK430) unbinding was 18.6 s(-1). Added 20 mM Pi slowed both monomer and dimer unbinding. With the roadblock in place, lead head attachment of dimers is prevented and ATP-induced trail head unbinding was then 42 s(-1). This is less than two-fold slower than the stepping rate of unimpeded rK430 dimers (50-70 s(-1)), indicating that during walking, lead head attachment induces at most only a slight (less than two-fold) acceleration of trail head detachment. As we discuss, this implies a coordination model having very fast (>2000 s(-1)) ATP-induced attachment of the lead head, followed by slower, strain-sensitive ADP release from the lead head.

Adenosine Diphosphate↗

PubMed2003

Kinetic analysis of Drosophila muscle myosin isoforms suggests a novel mode of mechanochemical coupling.

The molecular mechanism of myosin function was addressed by measuring transient kinetic parameters of naturally occurring and chimeric Drosophila muscle myosin isoforms. We assessed the native embryonic isoform, the native indirect flight muscle isoform, and two chimeric isoforms containing converter domains exchanged between the indirect flight muscle and embryonic isoforms. Myosin was purified from the indirect flight muscles of transgenic flies, and S1 was produced by alpha-chymotryptic digestion. Previous studies in vertebrate and scallop myosins have shown a correlation between actin filament velocity in motility assays and cross-bridge detachment rate, specifically the rate of ADP release. In contrast, our study showed no correlation between the detachment rate and actin filament velocity in Drosophila myosin isoforms and further that the converter domain does not significantly influence the biochemical kinetics governing the detachment of myosin from actin. We suggest that evolutionary pressure on a single muscle myosin gene may maintain a fast detachment rate in all isoforms. As a result, the attachment rate and completion of the power stroke or the equilibrium between actin.myosin.ADP states may define actin filament velocity for these myosin isoforms.

Actins↗

PubMed2003

Dynamic reorganization of the motor domain of myosin subfragment 1 in different nucleotide states.

Atomic models of the myosin motor domain with different bound nucleotides have revealed the open and closed conformations of the switch 2 element [Geeves, M.A. & Holmes, K.C. (1999) Annu. Rev. Biochem.68, 687-728]. The two conformations are in dynamic equilibrium, which is controlled by the bound nucleotide. In the present work we attempted to characterize the flexibility of the motor domain in the open and closed conformations in rabbit skeletal myosin subfragment 1. Three residues (Ser181, Lys553 and Cys707) were labelled with fluorophores and the probes identified three fluorescence resonance energy transfer pairs. The effect of ADP, ADP.BeFx, ADP.AlF4- and ADP.Vi on the conformation of the motor domain was shown by applying temperature-dependent fluorescence resonance energy transfer methods. The 50 kDa lower domain was found to maintain substantial rigidity in both the open and closed conformations to provide the structural basis of the interaction of myosin with actin. The flexibility of the 50 kDa upper domain was high in the open conformation and further increased in the closed conformation. The converter region of subfragment 1 became more rigid during the open-to-closed transition, the conformational change of which can provide the mechanical basis of the energy transduction from the nucleotide-binding pocket to the light-chain-binding domain.

Actins↗

PubMed2003

Probing nucleotide dissociation from myosin in vitro using microgram quantities of myosin.

The detailed kinetic analysis of novel myosin motors is often limited by the quantity of stable protein available for study. We show here that the use of coumarin based fluorescent ADP analogues allows the assay of ADP affinities and dissociation rate constants in a flash photolysis apparatus using microg quantities of the rabbit muscle myosin S1. We go on to use the analogues to characterise two other rat muscle myosin S1 and the motor domain of Dictyostelium cytoplasmic myosin II. The results show that the fluorescence change for the binding of a coumarin based ADP analogue to a myosin motor domain is variable in sign as well as amplitude for the different proteins. The analysis also provided estimates of the affinities of caged-ATP for S1 which were < or = 10 microM for muscle S1s and > 200 microM for the non-muscle myosin.

Adenosine Diphosphate↗

PubMed2002

A kinetic model of the co-operative binding of calcium and ADP to scallop (Argopecten irradians) heavy meromyosin.

Analysis of the kinetics of ATP and ADP binding to scallop (Argopecten irradians) heavy meromyosin (HMM) showed that the only calcium-dependent process is the rate of ADP release. At physiological ionic strength calcium accelerated ADP release about 20-fold. Notably in the absence of calcium only one ADP bound HMM, with an affinity of 0.5-1 microM. The second nucleotide site remained unoccupied at up to 50 microM ADP yet could bind ATP rapidly. The calcium dependence of ADP-release rates showed that calcium binds co-operatively to scallop HMM with an affinity of 0.78 microM and a Hill coefficient of 1.9. Detailed interpretation of the data suggests that HMM exists in equilibrium between the on and off states and that calcium and ADP modulate the equilibrium between the two states. The on state is favoured in the presence of calcium and in the absence of both calcium and nucleotide. The off state is favoured by ADP (or ADP * P(i)) in the absence of calcium. A detailed co-operative model of the interaction of ADP and calcium with HMM is presented.

Adenosine Diphosphate↗

PubMed2002

Intermonomer flexibility of Ca- and Mg-actin filaments at different pH values.

The fluorescence resonance energy transfer parameter, f, is defined as the efficiency of the energy transfer normalized by the quantum yield of the donor in the presence of acceptor. It is possible to characterize the flexibility of the protein matrix between the appropriate fluorescent probes by monitoring the temperature dependence of f. The intermonomer flexibility of the Ca-actin and Mg-actin filaments was characterized by using this method at pH values of 6.5 and 7.4. The protomers were labeled on Cys374 with donor [N-(((iodoacetyl)amino)ethyl)-5-naphthylamine-1-sulfonate; IAEDANS] or acceptor [5-(iodoacetamido)fluorescein; IAF] molecules. The temperature profile of f suggested that the intermonomer flexibility of actin filaments was larger at pH 7.4 than pH 6.5 in the case of Mg-F-actin while this difference was absent in the case of Ca-F-actin. More rigid intermonomer connection was identified at both pH values between the protomers of Mg-F-actin compared to the Ca-F-actin. The results were further supported by time dependent fluorescence measurements made on IAEDANS and IAF labeled Mg- and Ca-actin filaments at pH 6.5 and 7.4. Our spectroscopic results may suggest that the altered function of muscle following the change of pH within the muscle cells under physiological or pathological conditions might be affected by the modified dynamic properties of the magnesium saturated actin filaments. The change of the intracellular pH does not have an effect on the intermonomer flexibility of the Ca-actin filaments.

Actin Cytoskeleton↗