A Conformational Change in F-Actin When Myosin Binds:  Fluorescence Resonance Energy Transfer Detects an Increase in the Radial Coordinate of Cys-374

Moens, Pierre; Cg, dos Remedios

doi:10.1021/bi962588l

Cited by 30 publications

(17 citation statements)

References 39 publications

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“…the distance from the imaginary axis of a labeled side chain within the actin filament, where special symmetry conditions are fulfilled (1). The radial coordinate of a number of points in the actin filament and proximal relationships in the S1-and heavy meromyosin-decorated actin filament was determined by this approach (1,(17)(18)(19)(20)(21)(22)(23).Although FRET spectroscopy and x-ray crystallography are significantly different methods, there is usually a good correlation between the distances calculated from the atomic model and the ones obtained by . Accordingly, most of the FRET data obtained in the case of actin and acto-myosin are in a reasonably good agreement with the distances obtained by using the atomic models (5-7, 9).…”

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confidence: 99%

Conformational Distributions and Proximity Relationships in the Rigor Complex of Actin and Myosin Subfragment-1

Nyitrai¹,

Hild²,

Lukács³

et al. 2000

Journal of Biological Chemistry

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Cyclic conformational changes in the myosin head are considered essential for muscle contraction. We hereby show that the extension of the fluorescence resonance energy transfer method described originally by Taylor The cyclic interaction between actin and myosin is thought to provide the molecular basis for muscle contraction (2). There have been numerous attempts to characterize the molecular events underlying the contractile acto-myosin interaction (3). The recent availability of high resolution atomic structures of the actin monomer (4), the actin filament (5-7), myosin S1 (8), and the acto-S1 complex (9) provided a framework for the description of conformational changes associated with the contractile cycle.The atomic models permit the study of proximity relationships between various sites in the acto-myosin complex. An alternative method for studying proximal relationships within a macromolecule is FRET 1 spectroscopy. Several interprotein distances within the acto-myosin complex have been already determined with this latter method (10 -14). The distance between Cys-374 of actin and Cys-707 of S1 under rigor conditions was found to be 60 (11), 50 (12, 15), and 51 Å (14). By the use of scallop myosin hybrid molecules this distance was determined to be 45 Å (16). The distance between Cys-374 of actin and Cys-177 in the ELC of S1 in rigor was measured to be 50 (11) and 60 Å (10). FRET spectroscopy was applied to determine the radial coordinate, i.e. the distance from the imaginary axis of a labeled side chain within the actin filament, where special symmetry conditions are fulfilled (1). The radial coordinate of a number of points in the actin filament and proximal relationships in the S1-and heavy meromyosin-decorated actin filament was determined by this approach (1,(17)(18)(19)(20)(21)(22)(23).Although FRET spectroscopy and x-ray crystallography are significantly different methods, there is usually a good correlation between the distances calculated from the atomic model and the ones obtained by . Accordingly, most of the FRET data obtained in the case of actin and acto-myosin are in a reasonably good agreement with the distances obtained by using the atomic models (5-7, 9). However, an exception is the distance between Cys-177 on ELC of S1 and Cys-374 on the actin filament, because FRET revealed 50 -60 Å (10, 11), whereas the atomic model resulted in ϳ89 Å (9). Although this discrepancy may originate from biologically irrelevant factors (e.g. the nonzero size of the FRET probes (24)), the problem deserves further investigations because of the central role of the light chainbinding domain of S1 in the force generating process (9,27,28).In the present work we analyze the proximity relationships in the rigor acto-S1 complex, with a particular focus on the conformational distributions of the light chain-binding domain associated with a donor-acceptor distance distribution. The FRET method, developed originally by Taylor et al. (1) for radial coordinate determinations in actin filament, was extended to obtai...

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Conformational Distributions and Proximity Relationships in the Rigor Complex of Actin and Myosin Subfragment-1

Nyitrai¹,

Hild²,

Lukács³

et al. 2000

Journal of Biological Chemistry

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“…1b). In earlier studies this method was applied to determine the radial coordinate of a number of labeled amino acids in the actin filament (15)(16)(17)(18)(19)(20)(21)(22). FRET spectroscopy was also * This work was supported by the Hungarian Academy of Sciences, National Research Foundation OTKA Grants T032700, T023209, D32813, and F020174, Ministry of Education FKFP Grant 0463/99, and the Ministry of Health (202/99).…”

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“…1b). In earlier studies this method was applied to determine the radial coordinate of a number of labeled amino acids in the actin filament (15)(16)(17)(18)(19)(20)(21)(22). FRET spectroscopy was also used to characterize the flexibility of the protein matrix between a donor and an acceptor molecule (23) and to study the dynamic properties of the actin filament (24).…”

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confidence: 99%

Conformational and Dynamic Differences between Actin Filaments Polymerized from ATP- or ADP-Actin Monomers

Nyitrai

Hild

Hartvig

et al. 2000

Journal of Biological Chemistry

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Conformational and dynamic properties of actin filaments polymerized from ATP-or ADP-actin monomers were compared by using fluorescence spectroscopic methods. The fluorescence intensity of IAEDANS attached to the Cys 374 residue of actin was smaller in filaments from ADP-actin than in filaments from ATPactin monomers, which reflected a nucleotide-induced conformational difference in subdomain 1 of the monomer. Radial coordinate calculations revealed that this conformational difference did not modify the distance of Cys 374 from the longitudinal filament axis. Temperaturedependent fluorescence resonance energy transfer measurements between donor and acceptor molecules on Cys 374 of neighboring actin protomers revealed that the inter-monomer flexibility of filaments assembled from ADP-actin monomers were substantially greater than the one of filaments from ATP-actin monomers. Flexibility was reduced by phalloidin in both types of filaments.Actin is one of the most abundant proteins in biological systems and responsible for a number of cell functions in vivo (1, 2). Two principal forms of actin exist in living cells, the monomeric and the filamentous. Actins biological function depends on the actual dynamic and conformational properties of the protein and on the dynamic equilibrium between the two principal forms (1, 2).The polymerization of actin monomers with bound ATP is accompanied by the parallel hydrolysis of the nucleotide. However, the biological relevance of ATP hydrolysis by actin is still not well understood. Interestingly, ADP-monomeric actin also forms filaments (3), therefore, the presence of ATP and its hydrolysis are not essential for filament assembly. Previously, the hydrolysis of actin-bound ATP was assumed to play a key role in the steady-state treadmilling of actin filaments (4). Alternatively, Carlier (5, 6) suggested that ATP hydrolysis facilitated the rapid de-polymerization of actin.Recently, Janmey and colleagues (7) provided evidence that actin filaments polymerized from ATP-actin monomers were significantly stiffer than the ones obtained from ADP-monomers. Considering that both types of filaments consist mainly of ADP-actin protomers, this observation was explained by the assumption that the ATP-actin monomers were conformationally trapped following the hydrolysis of ATP, and the energy released during the hydrolysis was stored as elastic energy (7). The authors proposed that this elastic energy could play an important role when the actin filament interacted with actinbinding proteins. Although this exciting observation was later supported by other laboratories, a number of experimental results were contradictory.The direct effect of ATP on actin filaments was confirmed and extended to phalloidin-labeled actin by fluorescence microscopy experiments (8). Three-dimensional reconstructions from electron micrographs revealed similar effects of the nucleotides on the flexibility of actin filament (9, 10). The results of phosphorescence spectroscopic experiments apparently also supported the c...

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“…Supporting evidence lies in the fact that modification of the actin C-terminus by proteolysis, antibody binding and by mutagenesis can significantly impact actin polymerization properties [Johannes and Gallwitz, 1991;Crosbie et al, 1994;Orlova and Egelman, 1995;Phan et al 1997;Hegyi et al, 1998;Kim et al, 1998]. Attachment of fluorometric probes to different parts of the actin showed that polymerization per se and the binding of specific proteins to the actin filament at a distance from the probe site could affect probe behavior [Feng et al, 1997;Moens and dos Remedios, 1997;Wen et al, 2000;Bobkov et al, 2002Bobkov et al, , 2004Borovikov et al, 2004]. EM studies revealed that decoration of filaments by cofilin changed the filament twist [McGough et al, 1997;McGough and Chiu, 1999;Galkin et al, 2003Galkin et al, , 2011.…”

Section: Budding Yeast As An Expression System For Mutant Actinsmentioning

confidence: 99%

“…Rubenstein and Wen CYTOSKELETON specific proteins to the actin filament at a distance from the probe site could affect probe behavior [Feng et al, 1997;Moens and dos Remedios, 1997;Wen et al, 2000;Bobkov et al, 2002Bobkov et al, , 2004 Borovikov et al, 2004]. EM studies revealed that decoration of filaments by cofilin changed the filament twist [McGough et al, 1997;McGough and Chiu, 1999;Galkin et al, 2003Galkin et al, , 2011.…”

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Insights into the effects of disease‐causing mutations in human actins

Rubenstein

Wen

2014

Cytoskeleton

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Mutations in all six actins in humans have now been shown to cause diseases. However, a number of factors have made it difficult to gain insight into how the changes in actin functions brought about by these pathogenic mutations result in the disease phenotype. These include the presence of multiple actins in the same cell, limited accessibility to pure mutant material, and complexities associated with the structures and their component cells that manifest the diseases. To try to circumvent these difficulties, investigators have turned to the use of model systems. This review describes these various approaches, the initial results obtained using them, and the insight they have provided into allosteric mechanisms that govern actin function. Although results so far have not explained a particular disease phenotype at the molecular level, they have provided valuable insight into actin function at the mechanistic level which can be utilized in the future to delineate the molecular bases of these different actinopathies. Key Words: actin; allostery; disease; mutation; isoforms Introduction A ctin mutations can cause human disease. However, little is known concerning the mechanisms by which these mutations lead to the disease phenotypes they produce. The goal of this review is to describe the general properties of actin, the diseases associated with mutations in actin, methodologies that might be employed to achieve a mechanistic understanding of actin based disease and efforts that have been made toward this goal. Properties of G-and F-ActinA discussion of the effects of pathogenic mutations on actin function requires a basic understanding of actin structure and its solution properties. Actin is an abundant 42 kD monomeric protein, found in virtually all eukaryotic cells, that can cycle between either a monomeric (G-actin) or polymeric (F-actin) state. It plays both contractile and structural roles in the cytoplasm, and recent studies have documented that actin in the nucleus acts as a promoter of transcription or as a member of a number of chromatin remodeling complexes Philimonenko et al., 2004;Miyamoto and Gurdon, 2011; Miyamoto et al., 2011a,b;Weston et al., 2012].G-actin is a clam-shaped protein with two domains, the inner and outer domains ( Fig. 1), connected by a hinge region, and separated by a deep cleft. Outer (subdomains 1 and 2) and inner (subdomains 3 and 4) refer to their position in the actin filament. The N and C-termini reside on opposite faces of the protein in subdomain 1. An adenine nucleotide binds deep within the inter-domain cleft, held in place by a divalent cation, and imparts stability to the protein and the filament. This latter role depends on its ability to cycle between the ATP and ADP state by virtue of a polymerization-dependent ATPase activity [Carlier et al., 1987;Korn et al., 1987]. F-actin is a polar structure with pointed (2) and barbed (1) ends referring to the arrowhead pattern formed when myosin S1 binds to F-actin in the absence of ATP [Craig et al., 1985]. The polarity of th...

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A Conformational Change in F-Actin When Myosin Binds: Fluorescence Resonance Energy Transfer Detects an Increase in the Radial Coordinate of Cys-374

Cited by 30 publications

References 39 publications

Conformational Distributions and Proximity Relationships in the Rigor Complex of Actin and Myosin Subfragment-1

Conformational Distributions and Proximity Relationships in the Rigor Complex of Actin and Myosin Subfragment-1

Conformational and Dynamic Differences between Actin Filaments Polymerized from ATP- or ADP-Actin Monomers

Insights into the effects of disease‐causing mutations in human actins

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