Fluorescence resonance energy transfer within the complex formed by actin and myosin subfragment 1. Comparison between weakly and strongly attached states

Trayer, Hylary R.; Trayer, Ian P.

doi:10.1021/bi00415a049

Cited by 67 publications

(45 citation statements)

References 34 publications

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“…42. S1 fractionation into S1(A1) and S1(A2) isoenzymes was performed by ion-exchange chromatography on SP-Trisacryl M (43). Before use, samples of S1 were clarified by centrifugation at 300,000 ϫ g for 30 min.…”

Section: Methodsmentioning

confidence: 99%

The DNase-I Binding Loop of Actin May Play a Role in the Regulation of Actin-Myosin Interaction by Tropomyosin/Troponin

Moraczewska

Gruszczynska-Biegala

Redowicz

et al. 2004

Journal of Biological Chemistry

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, on its interaction with myosin S1 in the presence and absence of tropomyosin or tropomyosin-troponin. Both individual modifications reduced activation of S1 ATPase by actin to a similar extent. The effect of ECP cleavage, but not of subtilisin cleavage, was partially reversed by stabilization of interprotomer contacts with phalloidin, indicating different pathways of signal transmission from the N-and C-terminal parts of loop 38 -52 to myosin binding sites. ECP cleavage diminished the affinity to tropomyosin and reduced its inhibition of acto-S1 ATPase at low S1 concentrations, but increased the tropomyosin-mediated cooperative enhancement of the ATPase by S1 binding to actin. These effects were reversed by phalloidin. Subtilisin-cleaved actin more closely resembled unmodified actin than the ECP-modified actin. Limited proteolysis of the modified and unmodified F-actins revealed an allosteric effect of ECP cleavage on the conformation of the actin subdomain 4 region that is presumably involved in tropomyosin binding. Our results point to a possible role of the N-terminal part of loop 38 -52 of actin in communication between tropomyosin and myosin through changes in actin structure.

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Section: Methodsmentioning

confidence: 99%

The DNase-I Binding Loop of Actin May Play a Role in the Regulation of Actin-Myosin Interaction by Tropomyosin/Troponin

Moraczewska

Gruszczynska-Biegala

Redowicz

et al. 2004

Journal of Biological Chemistry

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“…Myosin S1 was prepared by chymotryptic digestion (which produces S1 lacking the RLC binding site but with an intact ELC and ELC-binding site) [12]. The isoenzymes, S1(A1) and S1(A2), were resolved on an SP-Trisacryl M (IBF Biotechnics) column [31]. Aliquots were shell frozen in the presence of 4 mg sucrose/mg S1 and 2 mM dithiothreitol and stored at Ϫ20°C.…”

Section: Methodsmentioning

confidence: 99%

“…Rabbit skeletal A1 light chain was prepared by urea dissociation from myosin as previously described [22]. Actin was labelled with N-(iodoacetyl) N′-(1-sulpho-5-napthyl) ethylenediamine (1,5-IAEDANS) as described in [31].…”

Section: Methodsmentioning

confidence: 99%

The N‐terminus of A1‐type myosin essential light chains binds actin and modulates myosin motor function

Timson

Trayer

1998

European Journal of Biochemistry

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There are two isoforms (A1 and A2) of the myosin essential light chain (ELC) and consequently two isoenzymes of myosin subfragment 1 (S1), S1(A1) and S1(A2). The two isoenzymes differ in their kinetic properties with S1(A1) having a lower apparent K m for actin and a slower turnover of MgATP (k cat ) than S1(A2). The two forms of the ELC differ only at their N-termini where A1 has an additional 40-odd amino acids that are not present in A2.The human atrial ELC (an A1-type ELC) was overexpressed in Escherichia coli and purified by ammonium sulphate fractionation and ion-exchange chromatography. The recombinant ELC had actinactivated MgATPase kinetics similar to those for rabbit skeletal S1(A1) under the same conditions. Deletion of the first 45 amino acid residues resulted in an ELC similar to the rabbit skeletal A2 isoform and, when hybridised into S1, in S1(A2)-like kinetic properties. Results obtained with an ELC mutant that lacks the first 11 residues were intermediate between these two extremes but tending towards the S1(A2)-like phenotype.The wild-type ELC (both hybridised into S1 or free in solution) could be cross-linked to F-actin, whereas the deletion mutant lacking the first 45 amino acids could not. The deletion mutant lacking the first 11 amino acids cross-linked only poorly under the same conditions, consistent with the MgATPase data. We therefore conclude that these N-terminal eleven amino acids predominantly encode an actinbinding site which modulates the kinetics of the myosin motor. Furthermore, while free A1-type ELC cross-linked to both polymeric F-actin and the monomeric G-actin:DNase-I complex, the same ELC in S1(A1) could only cross-link to F-actin. This suggests that the light chain binds to a different actin monomer than the heavy chain.

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“…S1 was prepared by chymotryptic digestion of myosin filaments (Weeds and Taylor, 1975) and purified over Sephacryl S-300 (Chaussepied et al, 1986). S1 isoenzymes containing alkali light chains 1 and 2 [Sl(Al) and Sl(A2) respectively] were separated by ion-exchange chromatography on SP-Trisacryl M (Trayer and Trayer, 1988). S1 was labeled on Cys707 (SH1) with I-AEDANS, as reported by Mornet et al (1981b).…”

Section: Proteinsmentioning

confidence: 99%

Photochemical cross‐linking of the skeletal myosin head heavy chain to actin subdomain‐1 at Arg95 and Arg28

Bonafé

Chaussepied

Capony

et al. 1993

European Journal of Biochemistry

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F-actin specifically substituted with the photocross-linker, p-azidophenylglyoxal, at Arg95 and Arg28 was isolated and characterized. Upon complexation with myosin subfragment-1 (Sl) and photolysis at 365 nm, it was readily cross-linked to the S1 heavy chain with a yield of about 13-25 %, generating four major actin-heavy-chain adducts with molecular masses in the range 165-240 kDa. The elevated Mg2'-ATPase of the covalent complexes displayed a turnover rate of 33 ? 8 s-I which is similar to the values reported earlier for other acto-S1 conjugates. The crosslinking between various proteolytic S1 and actin derivatives, combined with the fluorescent and immunochemical detection of the photocross-linked products, indicated that the arylnitrene group on Arg95 was inserted predominantly in the central 50-kDa region, whereas that attached to Arg28 mediated the selective cross-linking of the COOH-terminal 22-21-kDa fragments of the heavy chain, most probably by reacting at or near the connector segment between the 50-kDa and 20-kDa fragments. The rapid photoactivation and cross-linking to S1 of the substituted F-actin, which can be accomplished on a millisecond time scale, may serve to probe the structural dynamics of the interaction of the S1 heavy chain with subdomain-1 of actin during the ATPase cycle.During muscle contraction, the cyclic interactions between F-actin and the myosin heads or myosin subfragment-1 (Sl) are essential for the conversion of ATP hydrolysis into contractile force by the actomyosin-ATP complex. The nature of the nucleotide intermediates bound to the ATPase site is thought to determine the molecular movements and attitudes of the myosin heads over the actin filament (Botts et al., 1984;Huxley and Kress, 1985;Vibert and Cohen, 1988). The changes in the structure, localization and affinity of the binding sites at the actin-S1 interface would, therefore, play a major role in the transients of the displacement of S1 along F-actin.At present, it can be postulated that the generation of any acto-S 1 complex involves the specific association between the subdomain-1 of actin, the outermost domain of the actin protomere in the filament, containing all the known putative S1 interactive sites (Holmes et al., 1990;Milligan et al., 1990), and polypeptide stretches of the 20-kDa and 50-kDa fragments of the S1 heavy chain, the two potent actin-binding regions so far identified in the native protein (Mornet et Correspondence to P. Chaussepied or R.

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Fluorescence resonance energy transfer within the complex formed by actin and myosin subfragment 1. Comparison between weakly and strongly attached states

Cited by 67 publications

References 34 publications

The DNase-I Binding Loop of Actin May Play a Role in the Regulation of Actin-Myosin Interaction by Tropomyosin/Troponin

The DNase-I Binding Loop of Actin May Play a Role in the Regulation of Actin-Myosin Interaction by Tropomyosin/Troponin

The N‐terminus of A1‐type myosin essential light chains binds actin and modulates myosin motor function

Photochemical cross‐linking of the skeletal myosin head heavy chain to actin subdomain‐1 at Arg95 and Arg28

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