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KMID : 0613820040140050770
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Journal of Life Science
2004 Volume.14 No. 5 p.770 ~ p.777
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Functions of α-Tropomyosin Are Mainly Dependent upon the Local Structures of the Amino Terminus
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Cho Young-Jun
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Abstract
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It has been previously reported that unacetylated α-tropomyosin(TM) produced in E. coli failed to bind to actin while acetylated muscle TM and Ala-Ser dipeptide fusion TM (AS-TM) bound well to actin. In order to determine the structural requirement of the amino terminus for high actin affinity, a recombinant tropomyosin (Ala-TM) that a single Ala residue was added to the amino terminus of α-TM was constructed, overexpressed, and purified from E. coli. Actin affinity of Ala-TM was 2.3x106M-1, whereas that of unacetylated TM was considerably lower than 0.1x106M-1 indicating that addition of a single Ala residue to the amino terminus drastically increased, at least twenty times, actin affinity of TM. Ala-TM, however, bound to actin about three times weaker than acetylated TM and AS-TM, implying that the addition of an Ala residue was insufficient for complete restoration of high actin affinity. While Ala-TM, AS-TM, and muscle TM showed inhibition and activation of actomyosin S1 ATPase activity depending on myosin S1 concentration, the degree of inhibition and activation was different from each other. AS-TM exhibited the greatest inhibition of the ATPase at low S1 concentration, whereas the greatest activation of the ATPase was observed with muscle TM. These results, together with previous findings, strongly suggested that local structure of the amino terminus is the crucial functional determinant of TM.
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KEYWORD
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recombinant tropomyosins, actin binding, myosin S1 ATPase, N-acetylation
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