Subtilisin-cleaved actin: polymerization and interaction with myosin subfragment 1

Schwyter, Deborah H.; Phillips, Martin; Reisler, Emil

doi:10.1021/bi00440a027

Cited by 93 publications

(112 citation statements)

References 31 publications

(52 reference statements)

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“…However, addition of the bulky ABP does not abolish polymerizability, indicating that Gln-41 does not participate in actin-actin interactions crucial for polymer formation. The work of Schwyter et al (1989) has implicated residues near Gln-41 in actin-actin interactions as well as in S-1 binding. They cleaved actin at Met4'-Gly4*, and showed that the digested actin had a higher critical concentration for polymerization and a reduced affinity for myosin S-l .…”

Section: Actin-actin Contact Pointsmentioning

confidence: 99%

Gln‐41 is intermolecularly cross‐linked to Lys‐113 in F‐actin by N‐(4‐azidobenzoyl)‐putrescine

Hegyi¹,

Michel²,

Shabanowitz³

et al. 1992

Protein Science

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The bifunctional reagent N-(4-azidobenzoyl)-putrescine was synthesized and covalently bound to rabbit skeletal muscle actin. The incorporation was mediated by guinea pig liver transglutaminase under conditions similar to those described by Takashi (1988, Biochemistry 27, 938-943); up to 0.5 M/M were incorporated into G-actin, whereas F-actin was refractory to incorporation. Peptide fractionation showed that at least 90% of the label was bound to Gln-41. The labeled G-actin was polymerized, and irradiation of the F-actin led to covalent intermolecular cross-linking. A cross-linked peptide complex was isolated from a tryptic digest of the cross-linked actin in which digestion was limited to arginine; sequence analysis as well as mass spectrometry indicated that the linked peptides contained residues 40-62 and residues 96-116, and that the actual cross-link was between Gln-41 and Lys-113. Thus the y-carboxyl group of Gln-41 must be within 10.7 A of the side chain (probably the amino group) of Lys-I13 in an adjacent actin monomer. In the atomic model for F-actin proposed by Holmes et al. (1990, Nuture 347, 44-49), the a-carbons of these residues in adjacent monomers along the two-start helices are sufficiently close to permit cross-linking of their side chains, and, pending atomic resolution of the side chains, the results presented here seem to support the proposed model.

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Section: Actin-actin Contact Pointsmentioning

confidence: 99%

Gln‐41 is intermolecularly cross‐linked to Lys‐113 in F‐actin by N‐(4‐azidobenzoyl)‐putrescine

Hegyi¹,

Michel²,

Shabanowitz³

et al. 1992

Protein Science

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“…To delineate the MBS-substituted lysine mediating the cross-linking, we coupled S-l to subtilisin-cleaved MBS-G-actin. This protease was shown to clip native G-actin at Met-47 into 9 kDa Nterminal and 35 kDa C-terminal fragments which remain noncovalently associated [9]. The proteolytic MBS-actin derivative displayed an identical electrophoretic profile except that the 35 kDa peptide migrated as a doublet band reflecting the doublet composition of the parent MBS-actin (Fig.…”

Section: Localization Of the Cross-linking Site On The Actinmentioning

confidence: 94%

“…MBS-Gactin split at Met-47 with subtilisin was produced as described for All rights reserved. G-actin [9] by treatment of the actin derivative in G-buffer, pH 8.0 for 60 min at 25'C with subtilisin (protease to substrate weight ratio = 1:250).…”

Section: Chemicals and Protein Preparationsmentioning

confidence: 99%

The covalent maleimidobenzoyl‐actin‐myosin head complex

1994

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We have identified the region of actin involved in the covalent coupling of maleimidobenxoyl-G-actin to the central 50 kDa segment of the myosin-S-l heavy chain by analyzing the structure of the maleimidobenxoyl-G-actin-S-1 conjugate using selective proteolytic digestions, amino acid sequence determinations and novel cross-linking reactions between S-l and different maleimidobenxoyl-G-actin derivatives. The cross-linking is shown to occur only on the stretch of residues 4867 in actin subdomain-with Lys-50, residing on the outer part of the DNase-I-binding loop, as the most likely site of cross-linking. Because the maleimidobenxoyl-F-actin-S-1 complex undergoes the same coupling process, the data provide experimental evidence in favor of the recent model of the rigor F-a&in-S-l complex suggesting a close contact between structural elements of the lower domain of the 50 kDa fragment and the top of actin subdomain-2.Key worak F-actin; G-actin; Actomyosin interaction; Actin-myosin head cross-linking; Actin s&domain-2 IntroductloIlPreviously, we described the production and characterization of maleimidobenzoyl-G-actin (MBS-G-actin) and maleimidobenzoyl-F-actin (MBS-F-actin) [1,2]. The former derivative was generated by the reaction of skeletal monomeric actin with the heterobiftmctional agent, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MB-S), which resulted in the establishment of few intramolecular cross-links and the incorporation of an average of one maleimidobenzoyl group by the monofunctional acylation of a lysine residue of the protein. Although resistant to the salt-and myosin-S-l-induced polymerization, the MBS-G-actin could be readily converted into MBS-F-actin in the presence of phalloidin and MgCl, [2,3]. Both modified actins formed reversible and ATP-sensitive complexes with rabbit skeletal myosin-S-1 which were earlier investigated [ 1,3-51. Most importantly, the free maleimidobenzoyl group in the Gor F-MBS-actin was in a position that permits the actin subunit to couple covalently to the S-l heavy chain at the same region at which native F-actin binds to the heavy chain during rigor and the major covalent cross-link was shown to involve the central 50 kDa segment of the S-l heavy chain [1,2]. Hence, the covalent MBS-actinScomplex represents a useful tool for further assessing the molecular contacts between F-actin and myosin-S-l.In the present study we have identified the lysine of actin engaged in the MBS-promoted conjugation process by analysing the structure of the major 180 kDa adduct consisting of the MBS-actin molecule selectively bridged to the 95 kDa S-l heavy chain at the 50 kDa region [l]. The data show that the cross-linking takes place exclusively on the actin stretch of residues 48-67 and most likely involves Lys-50. This segment makes part of the actin subdomain-2. The findings directly demonstrate the spatial proximity of this subdomain to the S-l heavy chain in both complexes of S-l with G-or F-actin. They also provide an experimental support to the recent model of the rigor ...

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“…Although previous work from our laboratory (17) did not detect any subdomain 2 movements during regulation, all of our experiments used probes attached to Gln 41 (in ␣-skeletal actin) or Cys 41 and Cys 51 (in mutant yeast actins). Because such probes may have altered the dynamics, if not the conformation, of an intrinsically dynamic subdomain 2, and because TnI cross-links to Met 47 on actin, we tested for Tm/Tn interactions with this region using subtilisin-cleaved actin (3,18).…”

mentioning

confidence: 99%

“…Subtilisin cleaves between Met 47 and Gly 48 in loop 38 -52 of actin's subdomain 2 (3). The resultant protein exhibits modified interprotomer interactions in F-actin and impaired function with myosin (i.e.…”

mentioning

confidence: 99%

The Regulation of Subtilisin-cleaved Actin by Tropomyosin/Troponin

Pavlov¹,

Gerson²,

Yu³

et al. 2003

Journal of Biological Chemistry

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Vertebrate striated muscle contraction is regulated in a Ca 2؉ -dependent fashion by tropomyosin (Tm) and troponin (Tn). This regulation involves shifts in the position of Tm and Tn on actin filaments and may include conformational changes in actin that are then communicated to myosin subfragment 1 (S1). To determine whether subdomain 2 of actin plays a role in this regulation, the DNase-I loop 38 -52 of this subdomain was cleaved by subtilisin between residues Met 47 and Gly 48 . Despite impaired unregulated function, the potentiation and regulation of cleaved actin movement in the in vitro motility assay was not significantly different from that of uncleaved actin. Stopped-flow measurements of ADP release from regulated and unregulated cleaved acto-S1 showed a marked increase in ADP release from acto-S1 in the presence of the regulatory complex. The enhancement of the actin affinity for S1 in the presence of regulatory proteins was greater for uncleaved than for cleaved F-actin. Finally, both cleaved and uncleaved actins protect myosin loop 1 from papain cleavage equally well. Our results suggest that the potentiation of actin function in the in vitro motility assay by regulatory proteins stems from changes in cross-bridge cycle kinetics. In addition, the unimpaired calcium-sensitive regulation of cleaved actin indicates that subdomain 2 conformation does not play an essential role in the regulation process.

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Subtilisin-cleaved actin: polymerization and interaction with myosin subfragment 1

Cited by 93 publications

References 31 publications

Gln‐41 is intermolecularly cross‐linked to Lys‐113 in F‐actin by N‐(4‐azidobenzoyl)‐putrescine

Gln‐41 is intermolecularly cross‐linked to Lys‐113 in F‐actin by N‐(4‐azidobenzoyl)‐putrescine

The covalent maleimidobenzoyl‐actin‐myosin head complex

The Regulation of Subtilisin-cleaved Actin by Tropomyosin/Troponin

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