Role of the head-to-tail overlap region in smooth and skeletal muscle beta-tropomyosin.
Tropomyosin (Tm) is an alpha-helical, parallel, two-chain coiled coil which binds along the length of actin filaments in both muscle and non-muscle cells. Smooth and skeletal muscle Tms differ extensively at the C-terminus encoded by exon 9. Replacement of the striated muscle specific exon 9a-encoded C-terminus with that encoded by exon 9d expressed in smooth muscle and non-muscle cells increases the affinity of unacetylated alpha-SkTm for actin [Cho, Y. J., and Hitchcock-Degregori, S. E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10153-10157]. Here we show that swapping 10 amino acids at the C-terminus of beta-SkTm with the corresponding 10 amino acids of beta-SmTm had little effect on the regulation of S1 binding to actin, but Tm viscosity, Tm binding to actin, and troponin T1 binding to Tm all become like smooth rather than SkTm. beta-SkTm point mutations show that these properties are largely defined by the amino acids at two positions, 277 and 279. The N279L mutation reduces the viscosity of beta-SkTm to close to beta-SmTm values, while both residues contribute to the binding of TnT1. We also show that removing the first 11 N-terminal amino acids of beta-SmTm to make the mutant DeltaN-betaSmTm results in a 10-fold weakening in actin affinity compared to that of beta-SmTm. CD studies show no difference in thermal unfolding between beta-SmTm and DeltaN-betaSmTm; however, the viscosity of DeltaN-betaSmTm is much lower than that of the control. The results suggest that DeltaN-betaSmTm was unable to form filaments in solution but can form filaments on actin.
Coulton
Arthur T
AT
Department of Biosciences, University of Kent, Canterbury, UK.
Koka
Kezia
K
Lehrer
Sherwin S
SS
Geeves
Michael A
MA
D000199:Actins / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D005453:Fluorescence; D006801:Humans; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D014335:Tropomyosin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D000199:Actins; D020033:Protein Isoforms; D014335:Tropomyosin