Effect of nucleotides, actin and temperature on thermolysin digestion of myosin subfragment‐1

Mühlrád, András; Chaussepied, Natalie

doi:10.1111/j.1432-1033.1990.tb19273.x

Cited by 10 publications

(4 citation statements)

References 36 publications

(13 reference statements)

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“…We favour the second possibility because Lys553 is accessible also in the presence of actin as shown by the lack of actin effect on the nitromethane quenching of the fluorescence of the Lys553‐bound FH moiety and by the finding that the binding of FH‐S1 to F‐actin is not impeded [16]. Thus the present results support the earlier findings [14,15,28,29] that actin affects the conformation of myosin, which should be taken into account in describing the structural changes taking place during the cross‐bridge cycle.…”

Section: Discussionsupporting

confidence: 90%

Actin and nucleotide induced conformational changes in the vicinity of Lys553 in myosin subfragment 1

Peyser

Mühlrád

1999

European Journal of Biochemistry

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Biochemistry 34, 9500±9507] reported that 6-[fluoresceine-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS) selectively modifies Lys553, which is part of the strong actin-binding site of myosin subfragment 1 (S1). We found that the reaction of FHS with Lys533 is accompanied by a decrease in the fluorescence intensity of the reagent. The rate of the FHS reaction increased with increasing pH implying that the unprotonated form of the 1-amino group of Lys553 reacts with FHS. Addition of 0.4 m KCl reduced the rate of reaction significantly, which indicates ionic strengthdependent changes in the structure of S1. Limited trypsinolysis of S1 before the FHS reaction also decreased the rate of the reaction showing that the structural integrity of S1 is needed for the reactivity of Lys553. ATP, ADP, ADP´BeF x , ADP´AlF 4 , ADP´V i and pyrophosphate significantly decreased the rate of Lys553 labelling, suggesting nucleotide-induced conformational changes in the environment of Lys553. The fluorescence emission spectrum of the Lys553-bound FH moiety and the quenching of its fluorescence by nitromethane was not influenced by nucleotides, implying that the chemical reactivity but not the accessibility of Lys553 was decreased by the nucleotide-induced conformational change. In the presence of ATP when the M ** ADP´P i state of the ATPase cycle is predominantly populated, the reaction rate decreased more than in the case of the S1´ADP´AlF 4 2 and S1´ADP´V i complexes, which are believed to mimic the M ** ADP.P i state. This indicates that the conformation of the S1±ADP´AlF 4 2 and S1´ADP´V i complexes in the vicinity of Lys553 does not resemble the structure of the M ** ADP´P i state. The rate of Lys553 labelling decreased strongly in the presence of actin. The nitromethane quenching of the Lys553-bound FHS was not influenced by actin, which indicates that the reduced reaction rate is not due to steric hindrance caused by the bulky protein but by actin induced conformational changes in the vicinity of Lys553.

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Section: Discussionsupporting

confidence: 90%

Actin and nucleotide induced conformational changes in the vicinity of Lys553 in myosin subfragment 1

Peyser

Mühlrád

1999

European Journal of Biochemistry

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“…Thermolysin cleaves SI heavy chain at 26 kDa from the N-terminus (Applegate & Reisler, 1983) after Glu-208 or Ala-209 (Yamamoto, 1989b). This cleavage is enhanced by nucleotides and inhibited by actin (Applegate & Reisler, 1983Muhlrad & Chaussepied, 1990), indicating that communication exists between this site and both the nucleotideand actin-binding sites. We found that methylation does not affect the thermolysin cut in the absence of nucleotides and actin, except for a further degradation of the 26 kDa product.…”

Section: Resultsmentioning

confidence: 95%

Extensively Methylated Myosin Subfragment-1: Examination of Local Structure, Interactions with Nucleotides and Actin, and Ligand-Induced Conformational Changes

Cheung²,

Cj³

et al. 1994

Biochemistry

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The atomic structure of myosin subfragment-1 (S1) has been recently solved for crystals of extensively methylated S1 [Rayment et al. (1993) Science 261, 50-58]. In this study, the effect of such a modification on S1 structure and function was examined. According to the far- and near-ultraviolet CD spectra, the methylation does not affect the secondary structure of S1 but causes limited changes in its tertiary structure. The methylation significantly decreases the affinity of S1 for actin in rigor and, to a lesser degree, that of S1 to actin in the presence of MgATP gamma S. This modification, like the trinitrophenylation of Lys-83, accelerates the dissociation of a nucleotide trapped on S1 either by phosphate analogs or by cross-linking of the SH1 and SH2 thiols. Methylation strongly impairs the coupling between the actin- and nucleotide-binding sites as revealed by the reduced effect of actin on the release of epsilon ADP from the active site. It also causes a complete loss of in vitro motility of actin filaments over methylated HMM. In addition to this, methylation also impairs the communication between other sites on S1 including that between the nucleotide-binding site and SH1, and the actin-binding site and the 27/50 kDa junction and a site at 74 kDa from the N-terminus of S1. These changes are revealed in SH1 modification, thermolysin digestion, and vanadate-dependent photocleavage experiments, respectively. The increased rate of thermal denaturation of S1 and the loss of S1 protection by ADP and actin from this process also indicate flawed communications in methylated S1. It is concluded that these relatively mild but numerous and important changes impair the function of methylated S1.

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“…The former would suggest that the epitope is near to the myosin-actin interface, while the latter would indicate that there is communication between the actin and the antibody-binding sites on the 50 kDa domain. The first possibility is supported by the actin inhibition of the papain and the thermolysin cleavage at the 27 kDa/50 kDa junction of S-1 (Applegate & Reisler, 1983;Muhlrad & Chaussepied, 1989) while the second is supported by cross-linking studies (Morner et al, 1981;Sutoh, 1983) which point to the involvement of the C-terminal and not the N-terminal region of the 50 kDa fragment in the myosin-actin interface.…”

Section: Discussionmentioning

confidence: 96%

Localization of epitopes and functional effects of two novel monoclonal antibodies against skeletal muscle myosin

Dan-Goor

Silberstein

Kessel³

et al. 1990

J Muscle Res Cell Motil

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Two skeletal myosin monoclonal antibodies, raised against human skeletal myosin, were used to study the correlation between function, primary and tertiary structure of S-1 prepared from rabbit skeletal myosin. The heavy chain of S-1 is cleaved into three fragments by trypsin--27 kDa, 50 kDa and 20 kDa--aligned in this order from the N-terminus. The epitope of the first antibody was assigned to the N-terminal 1-23 amino acid stretch of S-1, since it reacted with the 27 kDa N-terminal tryptic fragment of S-1 but not with a derivative of the 27 kDa fragment, which lacks the above amino acid stretch. The epitope of the second antibody was assigned to the 3 kDa N-terminal region of the central 50 kDa domain of S-1. This assignment was based on proteolytic and photochemical cleavage of S-1 and on the labelling of its N-terminus by a specific antibody. The antibodies were visualized binding to the myosin head on electron micrographs of rotary-shadowed complexes of antibodies with myosin. Measurements on the micrographs indicated that the distances between the head-tail junction of myosin and the 'anti-27 K' and 'anti-50 K' epitopes are 14 nm and 17 nm, respectively. Both antibodies have a high affinity to S-1. The affinity of the 'anti-50 K' to S-1 decreased upon actin binding, while that of the 'anti-27 K' was not affected by binding of S-1 to F-actin. The 'anti-50 K' antibody inhibited the K+ (EDTA) and the actin-activated ATPase activity of S-1, while the 'anti-27 K' had no effect. The results indicate that either the epitope of the 'anti-50 K' is near to the actin or to the ATP-binding sites of S-1, or that there is communication, expressed as propagated conformational changes, between these sites and the epitope.

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Effect of nucleotides, actin and temperature on thermolysin digestion of myosin subfragment‐1

Cited by 10 publications

References 36 publications

Actin and nucleotide induced conformational changes in the vicinity of Lys553 in myosin subfragment 1

Actin and nucleotide induced conformational changes in the vicinity of Lys553 in myosin subfragment 1

Extensively Methylated Myosin Subfragment-1: Examination of Local Structure, Interactions with Nucleotides and Actin, and Ligand-Induced Conformational Changes

Localization of epitopes and functional effects of two novel monoclonal antibodies against skeletal muscle myosin

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