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.