Submicrosecond and microsecond rotational motions of myosin head in solution and in myosin synthetic filaments as revealed by time-resolved optical anisotropy decay measurements

Kazuhiko Kinosita, Shin'ichi Ishiwata, Hideyuki Yoshimura, Hiroshi Asai, Akira Ikegami

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

Rotational Brownian motions of the head portion (subfragment 1) of rabbit skeletal myosin were studied by the measurement of flash-induced absorption anisotropy decay and phosphorescence anisotropy decay of the triplet probe 5-eo-sinylmaleimide bound to the myosin head. Fluorescence anisotropy decay of the fluorescent probe N-(1-pyrenyl)male-imide was also examined in some cases. Most of the triplet signals were observed in the presence of 60% (w/w) sucrose, which simply reduced the rate of motion via viscosity damping, to obtain good time resolution. Anisotropy decay of eosin on isolated head fragment was single exponential over two decades; the data indicated that the largest diameter of the head was 14-17 nm if the head was modeled as a prolate ellipsoid of revolution and 12-13 nm if oblate. Anisotropy decay in myosin synthetic filaments consisted of a fast and a slow component and a small constant part; myosin monomers and heavy meromyosin gave similar but somewhat faster decays with a smaller residual anisotropy. For each sample, the decay curves between -10 and 30°C overlapped with each other after reducing the time scale to that at 20°C in the absence of sucrose, showing that no gross conformational changes occurred between these temperatures. The fast decay was in the submicrosecond range on the reduced time scale and could be explained by a wobbling motion of the head around the head-rod junction within a cone of semiangle 35° for filament and 41° for solubilized proteins. The slow decay had a relaxation time of a few microseconds and indicated that a part of the rod portion next to the head also wobbled extensively. Analysis in which the rod end was assumed to wobble uniformly in a cone suggested that the effective length of the wobbling part was about 14 nm, and the cone angle was estimated to be about 48° in filament and 57-60° for solubilized proteins.

Original languageEnglish
Pages (from-to)5963-5975
Number of pages13
JournalBiochemistry
Volume23
Issue number25
Publication statusPublished - 1984
Externally publishedYes

Fingerprint

Optical anisotropy
Anisotropy
Myosins
Head
Cones
Sucrose
Phosmet
Imides
Myosin Subfragments
Phosphorescence
Fluorescence Polarization
Brownian movement
Eosine Yellowish-(YS)
Fluorescent Dyes
Viscosity
Relaxation time
Proteins
Damping
Monomers
Fluorescence

ASJC Scopus subject areas

  • Biochemistry

Cite this

Submicrosecond and microsecond rotational motions of myosin head in solution and in myosin synthetic filaments as revealed by time-resolved optical anisotropy decay measurements. / Kinosita, Kazuhiko; Ishiwata, Shin'ichi; Yoshimura, Hideyuki; Asai, Hiroshi; Ikegami, Akira.

In: Biochemistry, Vol. 23, No. 25, 1984, p. 5963-5975.

Research output: Contribution to journalArticle

Kinosita, Kazuhiko ; Ishiwata, Shin'ichi ; Yoshimura, Hideyuki ; Asai, Hiroshi ; Ikegami, Akira. / Submicrosecond and microsecond rotational motions of myosin head in solution and in myosin synthetic filaments as revealed by time-resolved optical anisotropy decay measurements. In: Biochemistry. 1984 ; Vol. 23, No. 25. pp. 5963-5975.
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N2 - Rotational Brownian motions of the head portion (subfragment 1) of rabbit skeletal myosin were studied by the measurement of flash-induced absorption anisotropy decay and phosphorescence anisotropy decay of the triplet probe 5-eo-sinylmaleimide bound to the myosin head. Fluorescence anisotropy decay of the fluorescent probe N-(1-pyrenyl)male-imide was also examined in some cases. Most of the triplet signals were observed in the presence of 60% (w/w) sucrose, which simply reduced the rate of motion via viscosity damping, to obtain good time resolution. Anisotropy decay of eosin on isolated head fragment was single exponential over two decades; the data indicated that the largest diameter of the head was 14-17 nm if the head was modeled as a prolate ellipsoid of revolution and 12-13 nm if oblate. Anisotropy decay in myosin synthetic filaments consisted of a fast and a slow component and a small constant part; myosin monomers and heavy meromyosin gave similar but somewhat faster decays with a smaller residual anisotropy. For each sample, the decay curves between -10 and 30°C overlapped with each other after reducing the time scale to that at 20°C in the absence of sucrose, showing that no gross conformational changes occurred between these temperatures. The fast decay was in the submicrosecond range on the reduced time scale and could be explained by a wobbling motion of the head around the head-rod junction within a cone of semiangle 35° for filament and 41° for solubilized proteins. The slow decay had a relaxation time of a few microseconds and indicated that a part of the rod portion next to the head also wobbled extensively. Analysis in which the rod end was assumed to wobble uniformly in a cone suggested that the effective length of the wobbling part was about 14 nm, and the cone angle was estimated to be about 48° in filament and 57-60° for solubilized proteins.

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