Cross-bridge orientation in skeletal muscle measured by linear dichroism of an extrinsic chromophore

Julian Borejdo, Olga Assulin, Toshio Ando, Susan Putnam

Research output: Contribution to journalArticle

86 Citations (Scopus)

Abstract

Linear dichroism of chromophoric labels attached to myosin heads has been used to establish cross-bridge orientation in myofibrils and muscle fibers. Generalized expressions were obtained for the dichroic ratio of a circularly symmetrical assembly of chromophores viewed through high apertures. The theoretical expressions were used to estimate the angle Θ of the absorption dipole of the dye relative to the myofibrillar axis. Myosin subfragment-1 has been labeled with tetramethyl rhodamine and diffused into the I-band of myofibrils; endogenous muscle myosin has been labeled directly. Dichroism has been measured from these preparations in the absence (rigor) and presence of MgATP and its analogs. In rigor, angle Θ was 80 °. Relaxed and contracted preparations displayed no dichroism, suggesting a high degree of cross-bridge disorder. MgAMP-PNP † Abbreviations used: MgAMP-PNP, Mg-5′-adenylylimidodiphosphate; AP5A, P1, P5-diadenosine-5′-pentaphosphate; IATR, iodoacetamidotetramethylrhodamine; IAF, 5-iodoacetamidofluorescein; 1,5-IAEDANS, 5-iodoacetamidoethylaminonaphthalene-1-sulfonic acid; HMM, heavy meromyosin; S-1, myosin subfragment-1; TRFAD, time-resolved fluorescence anisotropy decay; EGTA, ethyleneglycolbis(β-aminoethyl ether)N,N′-tetraacetic acid. and MgPPi imposed on the cross-bridges a distribution intermediate between rigor and relaxation. In the presence of MgADP the preparations showed strong dichroism of the opposite direction to that present in rigor. No detachment of the cross-bridges occurred under these conditions and the effect was not due to the rotational displacement of the attached dye by the nucleotide. It is concluded that the formation of a ternary complex myosin-MgADP-actin makes it possible to detect a large local deformation imposed on the cross-bridge by nucleotide binding, which results in a change of the spatial attitude of the mobile region of the protein by about 40 °.

Original languageEnglish
Pages (from-to)391-411
Number of pages21
JournalJournal of Molecular Biology
Volume158
Issue number3
DOIs
StatePublished - 5 Jul 1982

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Myosin Subfragments
Myosins
Skeletal Muscle
Myofibrils
Adenosine Diphosphate
Coloring Agents
Nucleotides
Adenylyl Imidodiphosphate
Muscles
Fluorescence Polarization
Rhodamines
Sulfonic Acids
Egtazic Acid
Ether
Actins
Adenosine Triphosphate
Acids
Proteins

Cite this

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title = "Cross-bridge orientation in skeletal muscle measured by linear dichroism of an extrinsic chromophore",
abstract = "Linear dichroism of chromophoric labels attached to myosin heads has been used to establish cross-bridge orientation in myofibrils and muscle fibers. Generalized expressions were obtained for the dichroic ratio of a circularly symmetrical assembly of chromophores viewed through high apertures. The theoretical expressions were used to estimate the angle Θ of the absorption dipole of the dye relative to the myofibrillar axis. Myosin subfragment-1 has been labeled with tetramethyl rhodamine and diffused into the I-band of myofibrils; endogenous muscle myosin has been labeled directly. Dichroism has been measured from these preparations in the absence (rigor) and presence of MgATP and its analogs. In rigor, angle Θ was 80 °. Relaxed and contracted preparations displayed no dichroism, suggesting a high degree of cross-bridge disorder. MgAMP-PNP† † Abbreviations used: MgAMP-PNP, Mg-5′-adenylylimidodiphosphate; AP5A, P1, P5-diadenosine-5′-pentaphosphate; IATR, iodoacetamidotetramethylrhodamine; IAF, 5-iodoacetamidofluorescein; 1,5-IAEDANS, 5-iodoacetamidoethylaminonaphthalene-1-sulfonic acid; HMM, heavy meromyosin; S-1, myosin subfragment-1; TRFAD, time-resolved fluorescence anisotropy decay; EGTA, ethyleneglycolbis(β-aminoethyl ether)N,N′-tetraacetic acid. and MgPPi imposed on the cross-bridges a distribution intermediate between rigor and relaxation. In the presence of MgADP the preparations showed strong dichroism of the opposite direction to that present in rigor. No detachment of the cross-bridges occurred under these conditions and the effect was not due to the rotational displacement of the attached dye by the nucleotide. It is concluded that the formation of a ternary complex myosin-MgADP-actin makes it possible to detect a large local deformation imposed on the cross-bridge by nucleotide binding, which results in a change of the spatial attitude of the mobile region of the protein by about 40 °.",
author = "Julian Borejdo and Olga Assulin and Toshio Ando and Susan Putnam",
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language = "English",
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Cross-bridge orientation in skeletal muscle measured by linear dichroism of an extrinsic chromophore. / Borejdo, Julian; Assulin, Olga; Ando, Toshio; Putnam, Susan.

In: Journal of Molecular Biology, Vol. 158, No. 3, 05.07.1982, p. 391-411.

Research output: Contribution to journalArticle

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AU - Borejdo, Julian

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N2 - Linear dichroism of chromophoric labels attached to myosin heads has been used to establish cross-bridge orientation in myofibrils and muscle fibers. Generalized expressions were obtained for the dichroic ratio of a circularly symmetrical assembly of chromophores viewed through high apertures. The theoretical expressions were used to estimate the angle Θ of the absorption dipole of the dye relative to the myofibrillar axis. Myosin subfragment-1 has been labeled with tetramethyl rhodamine and diffused into the I-band of myofibrils; endogenous muscle myosin has been labeled directly. Dichroism has been measured from these preparations in the absence (rigor) and presence of MgATP and its analogs. In rigor, angle Θ was 80 °. Relaxed and contracted preparations displayed no dichroism, suggesting a high degree of cross-bridge disorder. MgAMP-PNP† † Abbreviations used: MgAMP-PNP, Mg-5′-adenylylimidodiphosphate; AP5A, P1, P5-diadenosine-5′-pentaphosphate; IATR, iodoacetamidotetramethylrhodamine; IAF, 5-iodoacetamidofluorescein; 1,5-IAEDANS, 5-iodoacetamidoethylaminonaphthalene-1-sulfonic acid; HMM, heavy meromyosin; S-1, myosin subfragment-1; TRFAD, time-resolved fluorescence anisotropy decay; EGTA, ethyleneglycolbis(β-aminoethyl ether)N,N′-tetraacetic acid. and MgPPi imposed on the cross-bridges a distribution intermediate between rigor and relaxation. In the presence of MgADP the preparations showed strong dichroism of the opposite direction to that present in rigor. No detachment of the cross-bridges occurred under these conditions and the effect was not due to the rotational displacement of the attached dye by the nucleotide. It is concluded that the formation of a ternary complex myosin-MgADP-actin makes it possible to detect a large local deformation imposed on the cross-bridge by nucleotide binding, which results in a change of the spatial attitude of the mobile region of the protein by about 40 °.

AB - Linear dichroism of chromophoric labels attached to myosin heads has been used to establish cross-bridge orientation in myofibrils and muscle fibers. Generalized expressions were obtained for the dichroic ratio of a circularly symmetrical assembly of chromophores viewed through high apertures. The theoretical expressions were used to estimate the angle Θ of the absorption dipole of the dye relative to the myofibrillar axis. Myosin subfragment-1 has been labeled with tetramethyl rhodamine and diffused into the I-band of myofibrils; endogenous muscle myosin has been labeled directly. Dichroism has been measured from these preparations in the absence (rigor) and presence of MgATP and its analogs. In rigor, angle Θ was 80 °. Relaxed and contracted preparations displayed no dichroism, suggesting a high degree of cross-bridge disorder. MgAMP-PNP† † Abbreviations used: MgAMP-PNP, Mg-5′-adenylylimidodiphosphate; AP5A, P1, P5-diadenosine-5′-pentaphosphate; IATR, iodoacetamidotetramethylrhodamine; IAF, 5-iodoacetamidofluorescein; 1,5-IAEDANS, 5-iodoacetamidoethylaminonaphthalene-1-sulfonic acid; HMM, heavy meromyosin; S-1, myosin subfragment-1; TRFAD, time-resolved fluorescence anisotropy decay; EGTA, ethyleneglycolbis(β-aminoethyl ether)N,N′-tetraacetic acid. and MgPPi imposed on the cross-bridges a distribution intermediate between rigor and relaxation. In the presence of MgADP the preparations showed strong dichroism of the opposite direction to that present in rigor. No detachment of the cross-bridges occurred under these conditions and the effect was not due to the rotational displacement of the attached dye by the nucleotide. It is concluded that the formation of a ternary complex myosin-MgADP-actin makes it possible to detect a large local deformation imposed on the cross-bridge by nucleotide binding, which results in a change of the spatial attitude of the mobile region of the protein by about 40 °.

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