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.
|Number of pages||13|
|Publication status||Published - 1984|
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