The mechanism of force generation in myosin

A disorder-to-order transition, coupled to internal structural changes

D. D. Thomas, S. Ramachandran, O. Roopnarine, D. W. Hayden, E. M. Ostap, I. Rayment, K. Reisler, K. Kinosita

Research output: Contribution to journalArticle

82 Citations (Scopus)

Abstract

We propose a molecular mechanism of force generation in muscle, based primarily on site-specific spectroscopic probe studies of myosin heads in contracting muscle fibers and myofibrils. Electron paramagnetic resonance (EPR) and time-resolved phosphorescence anisotropy (TPA) of probes attached to SH1 (Cys 707, in the catalytic domain of the head) have consistently shown that most myosin heads in contracting muscle are dynamically disordered, undergoing large-amplitude rotations in the μs time range. Some of these disordered heads are bound to actin, especially in the early (weak-binding, preforce) phase of the ATPase cycle. The small ordered population (10-20%) is rigidly oriented precisely as in rigor, with no other distinct angle observed in contraction or in the presence of intermediate states trapped by nucleotide analogs. These results are not consistent with the classical model in which the entire head undergoes a 45° transition between two distinct orientations. Therefore, it has been proposed that the catalytic domain of the myosin head has only one stereospecific (rigor-like) actin-binding angle, and that the head's internal structure changes during force generation, causing the distal light-chain-binding domain to rotate. To test this model, we have performed EPR and TPA studies of probes attached to regulatory light chains (RLCs) in rabbit and scallop myofibrils and fibers. The RLC results confirm the predominance of dynamic (μs) rotational disorder in both relaxation and contraction, and show that the different mechanisms of calcium regulation in the two muscles produce different rotational dynamics. In rabbit myofibrils, RLC probes are more dynamically disordered than SH1 probes, especially in rigor and contraction, indicating that the light- chain-binding domain undergoes rotational motions relative to the catalytic domain when myosin heads interact with actin. An SH1-bound spin label, which is sensitive to myosin's internal dynamics, resolves three distinct conformations during contraction, and time-resolved EPR shows that these transitions are coupled to specific steps in the ATPase cycle. We propose that force is generated during contraction by a disorder-to-order transition, in which myosin heads first attach weakly to actin in a nonstereospecific mode characterized by large-scale dynamic disorder, then undergo at least two conformational transitions involving large-scale structural (rotational) changes within the head, culminating in a highly ordered strong-binding state that bears force.

Original languageEnglish
JournalBiophysical Journal
Volume68
Issue number4 SUPPL.
Publication statusPublished - 1995
Externally publishedYes

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Nonmuscle Myosin Type IIA
Myosins
Head
Myofibrils
Actins
Electron Spin Resonance Spectroscopy
Light
Muscles
Catalytic Domain
Anisotropy
Adenosine Triphosphatases
Pectinidae
Rabbits
Spin Labels
Nucleotides
Calcium

ASJC Scopus subject areas

  • Biophysics

Cite this

Thomas, D. D., Ramachandran, S., Roopnarine, O., Hayden, D. W., Ostap, E. M., Rayment, I., ... Kinosita, K. (1995). The mechanism of force generation in myosin: A disorder-to-order transition, coupled to internal structural changes. Biophysical Journal, 68(4 SUPPL.).

The mechanism of force generation in myosin : A disorder-to-order transition, coupled to internal structural changes. / Thomas, D. D.; Ramachandran, S.; Roopnarine, O.; Hayden, D. W.; Ostap, E. M.; Rayment, I.; Reisler, K.; Kinosita, K.

In: Biophysical Journal, Vol. 68, No. 4 SUPPL., 1995.

Research output: Contribution to journalArticle

Thomas, DD, Ramachandran, S, Roopnarine, O, Hayden, DW, Ostap, EM, Rayment, I, Reisler, K & Kinosita, K 1995, 'The mechanism of force generation in myosin: A disorder-to-order transition, coupled to internal structural changes', Biophysical Journal, vol. 68, no. 4 SUPPL..
Thomas DD, Ramachandran S, Roopnarine O, Hayden DW, Ostap EM, Rayment I et al. The mechanism of force generation in myosin: A disorder-to-order transition, coupled to internal structural changes. Biophysical Journal. 1995;68(4 SUPPL.).
Thomas, D. D. ; Ramachandran, S. ; Roopnarine, O. ; Hayden, D. W. ; Ostap, E. M. ; Rayment, I. ; Reisler, K. ; Kinosita, K. / The mechanism of force generation in myosin : A disorder-to-order transition, coupled to internal structural changes. In: Biophysical Journal. 1995 ; Vol. 68, No. 4 SUPPL.
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T2 - A disorder-to-order transition, coupled to internal structural changes

AU - Thomas, D. D.

AU - Ramachandran, S.

AU - Roopnarine, O.

AU - Hayden, D. W.

AU - Ostap, E. M.

AU - Rayment, I.

AU - Reisler, K.

AU - Kinosita, K.

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N2 - We propose a molecular mechanism of force generation in muscle, based primarily on site-specific spectroscopic probe studies of myosin heads in contracting muscle fibers and myofibrils. Electron paramagnetic resonance (EPR) and time-resolved phosphorescence anisotropy (TPA) of probes attached to SH1 (Cys 707, in the catalytic domain of the head) have consistently shown that most myosin heads in contracting muscle are dynamically disordered, undergoing large-amplitude rotations in the μs time range. Some of these disordered heads are bound to actin, especially in the early (weak-binding, preforce) phase of the ATPase cycle. The small ordered population (10-20%) is rigidly oriented precisely as in rigor, with no other distinct angle observed in contraction or in the presence of intermediate states trapped by nucleotide analogs. These results are not consistent with the classical model in which the entire head undergoes a 45° transition between two distinct orientations. Therefore, it has been proposed that the catalytic domain of the myosin head has only one stereospecific (rigor-like) actin-binding angle, and that the head's internal structure changes during force generation, causing the distal light-chain-binding domain to rotate. To test this model, we have performed EPR and TPA studies of probes attached to regulatory light chains (RLCs) in rabbit and scallop myofibrils and fibers. The RLC results confirm the predominance of dynamic (μs) rotational disorder in both relaxation and contraction, and show that the different mechanisms of calcium regulation in the two muscles produce different rotational dynamics. In rabbit myofibrils, RLC probes are more dynamically disordered than SH1 probes, especially in rigor and contraction, indicating that the light- chain-binding domain undergoes rotational motions relative to the catalytic domain when myosin heads interact with actin. An SH1-bound spin label, which is sensitive to myosin's internal dynamics, resolves three distinct conformations during contraction, and time-resolved EPR shows that these transitions are coupled to specific steps in the ATPase cycle. We propose that force is generated during contraction by a disorder-to-order transition, in which myosin heads first attach weakly to actin in a nonstereospecific mode characterized by large-scale dynamic disorder, then undergo at least two conformational transitions involving large-scale structural (rotational) changes within the head, culminating in a highly ordered strong-binding state that bears force.

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