Requirement of Amino-terminal Modification for Striated Muscle α-Tropomyosin Function

Striated muscle α-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 α-tropomyosin was expressed in insect Sf9 cells using the baculovirus expression vector system. The purified tropomyosin (∼15 mg/liter of insect cell suspension) polymerized, comigrated with chicken striated α-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 α-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 α-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.