Nucleutide- and temperature- induced changes in myosin subfragment-1 structure

doi:10.1016/0167-4838(92)90055-I

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 1159, Issue 3, 20 October 1992, Pages 267-273

Biochimica et Biophy...

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Abstract

The effects of nucleotide binding and temperature on the internal structural dynamics of myosin subfragment 1 (S1) were monitored by intrinsic tryptophan phosphorescence lifetime and fluorescence anisotropy measurements. Changes in the global conformation of S1 were monitored by measuring its rate of rotational diffusion using transient electric birefringence techniques. At 5°C, the binding of MgADP, MgADP,P and MgADP,V (vanadate) progressively reduce the rotational freedom of S1 tryptophans, producing what appear to be increasingly more rigidified S1-nucleotide structures. The changes in the luminescence properties of the tryptophans suggest that at least one is located at the interface of two S1 subdomains. Increasing the temperature from 0 to 25°C increases the apparent internal mobility of S1 tryptophans in all cases and, in addition, a reversible temperature-dependent transition centered near 15°C was observed for S1,S1-MgADP and S1-MgADP,P, but not for S1-MgADP,V. The rotational diffusion constants of S1 and S1-MgADP were measured at temperatures between 0 and 25°C. After adjusting for the temperature and viscosity of the solvent, the data indicate that the thermally induced transition at 15°C comprises local conformational changes, but no global conformational change. Structural features of S1-MgADP,P, which may relate to its role in force generation while bound to actin, are presented.

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Citation Excerpt :
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It is well established that in a skeletal muscle under relaxing conditions, cross-bridges exist in a mixture of four weak binding states in equilibrium (A·M·ATP, A·M·ADP·P_i, M·ATP, and M·ADP·P_i). It has been shown that these four weak binding states are in the pathway to force generation. In the past their structural, biochemical, and mechanical properties have been characterized as a group. However, it was shown that the myosin heads in the M·ATP state exhibited a disordered distribution along the thick filament, while in the M·ADP·P_i state they were well ordered. It follows that the structures of the weakly attached states of A·M·ATP and A·M·ADP·P_i could well be different. Individual structures of the two attached states could not be assigned because protocol for isolating the two states has not been available until recently. In the present study, muscle fibers are reacted with N-phenylmaleimide such that ATP hydrolysis is inhibited, i.e., the cross-bridge population under relaxing conditions is distributed only between the two states of M·ATP and A·M·ATP. Two-dimensional x-ray diffraction was applied to determine the structural characteristics of the attached A·M·ATP state. Because the detached state of M·ATP is disordered and does not contribute to layer line intensities, changes as a result of increasing attachment in the A·M·ATP state are attributable to that state alone. The equilibrium toward the attached state was achieved by lowering the ionic strength. The results show that upon attachment, both the myosin and the first actin associated layer lines increased intensities, while the sixth actin layer line was not significantly affected. However, the intensities remain weak despite substantial attachment. The results, together with modeling (see J. Gu, S. Xu and L. C. Yu, 2002, Biophys. J. 82:2123–2133),suggest that there is a wide range of orientation of the attached A·M·ATP cross-bridges while the myosin heads maintain some degree of helical distribution on the thick filament, suggesting a high degree of flexibility in the actomyosin complex. Furthermore, the lack of sensitivity of the sixth actin layer line suggests that the binding site on actin differs from the putative site for rigor binding. The significance of the flexibility in the A·M·ATP complex in the process of force generation is discussed.
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Nucleutide- and temperature- induced changes in myosin subfragment-1 structure

Abstract

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol Chem.

Biophys. J.

Trends Biochem. Sci.

Methods Enzymol.

J. Mol. Biol.

Methods Enzymol.

Arch. Biochem. Biopys.

Biochemistry

Science

Annu. Rev. Phys. Chem.

Annu. Rev. Physiol.

Biochem. J.

Biochemistry

Biochemistry

Biochemistry

Biochem.

J. Biochem.

Biochemistry

Biochemistry

Biochemistry

Biochemistry

Eur. J. Biochem.

Science

Biochemistry

J. Phys. Chem.

Eur. J. Biochem.