Requirement of amino-terminal modification for striated muscle alpha-tropomyosin function.
Striated muscle alpha-tropomyosin expressed in Escherichia coli is unacetylated, polymerizes poorly, and binds weakly to F-actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). To define the structural requirements of NH2-terminal modification for striated tropomyosin function, an acetylated recombinant tropomyosin and an unacetylated short fusion recombinant tropomyosin were compared. An acetylated recombinant chicken striated muscle alpha-tropomyosin was expressed in insect Sf9 cells using the baculovirus expression vector system. The purified tropomyosin (approximately 15 mg/liter of insect cell suspension) polymerized, comigrated with chicken striated alpha-tropomyosin purified from muscle on two-dimensional polyacrylamide gels, was blocked at the NH2 terminus, and had the same actin affinity as muscle tropomyosin. These results conclusively show the importance of NH2-terminal acetylation for striated tropomyosin function. To learn if a short fusion peptide would substitute for amino-terminal acetylation, tropomyosin with AlaSer-Arg on the NH2 terminus was constructed and expressed in E. coli as an unacetylated protein. This f3-tropomyosin bound to actin with a 10-fold higher affinity than striated muscle alpha-TM and, unlike muscle tropomyosin, exhibited a shear-dependent viscosity. The altered function of f3-tropomyosin shows that the naturally occurring acetylated NH2 terminus is required for full, normal function. It is proposed that a major requirement for cooperative binding of striated muscle tropomyosin to actin is modification of the alpha-amino group of methionine to be an amide, as when acetylated or in a peptide bond in a fusion protein, to make the extreme NH2 terminus more hydrophobic. The results are discussed in terms of known coiled coil structure.
Urbancikova
M
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Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
Hitchcock-DeGregori
S E
SE
D000107:Acetylation; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D009036:Moths; D018482:Muscle, Skeletal / Q000502:physiology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D014335:Tropomyosin / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
D000199:Actins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D014335:Tropomyosin