The effect of single residue substitutions of serine-283 on the strength of head-to-tail interaction and actin binding properties of rabbit skeletal muscle alpha-tropomyosin.
Vertebrate skeletal muscle alpha-tropomyosin polymerizes in a head-to-tail manner and binds cooperatively to actin. It has been postulated that the cooperative actin binding is governed by the strength of the head-to-tail interaction. In order to know the relationship between the head-to-tail affinity and actin binding, we studied the properties of tropomyosin variants with single residue substitutions at serine-283, the penultimate residue at the carboxyl terminus that is involved in the head-to-tail interaction. It has been shown that the phosphorylation of serine-283 strengthens the head-to-tail interaction. Viscometry was employed to compare the head-to-tail affinity of tropomyosin variants. Variant S283E showed higher viscosity whereas variant S283K showed lower viscosity compared with the wild type non-phosphorylated alpha-tropomyosin. The results confirm the idea that the interaction is sensitive to the ionic properties of residue 283. The strength of the head-to-tail interaction was assessed directly by sedimentation equilibrium using two pairs of tropomyosin variants designed so that only dimeric interactions were allowed within each pair. From one pair of variants with serine-283, the association constant was determined to be 2.6 x 10(4) M(-1) (SD =1.0 x 10(4)), whereas for the second pair with glutamate-283, the affinity was 3.9 x 10(4) M(-1) (SD =1.6 x 10(4)), slightly stronger than the former, consistent with the results of viscometry. The results indicate that the head-to-tail association is weak as previously implicated from light scattering measurements. Cosedimentation was employed to measure the cooperative actin binding of tropomyosin variants. Although previous results indicated the phosphorylation has no significant influence on the actin affinity, variant S283E shows a lower affinity compared with the control. Variants S283K and S283A show even lower affinities to actin, although these species bind to actin more cooperatively than does variant S283E. The results indicate that the affinity of the head-to-tail interaction between adjacent tropomyosin molecules is weak, and is substantially influenced by an extra charge at residue 283. On the other hand, the interaction with actin, the affinity and the cooperativity in actin binding, is dependent on amino acid residues at 283 and is not simply correlated with the strength of the head-to-tail interaction between Tm molecules in solution.
Sano
K
K
International Institute for Advanced Research, Central Research Laboratories, Matsushita Electric Industrial, Hikari-dai, Seika, Kyoto 619-0237, Japan.
Maeda
K
K
Oda
T
T
Maéda
Y
Y
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002942:Circular Dichroism; D017931:DNA Primers; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D007313:Insecta / Q000235:genetics; D018482:Muscle, Skeletal / Q000737:chemistry; D017354:Point Mutation; D011817:Rabbits; D012694:Serine / Q000235:genetics*; D014335:Tropomyosin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D014461:Ultracentrifugation; D014783:Viscosity
D000199:Actins; D017931:DNA Primers; D014335:Tropomyosin; D012694:Serine