Sano K, Maeda K, Oda T, Maéda Y
International Institute for Advanced Research, Central Research Laboratories, Matsushita Electric Industrial, Hikari-dai, Seika, Kyoto 619-0237, Japan.
J Biochem. 2000 Jun;127(6):1095-102. doi: 10.1093/oxfordjournals.jbchem.a022703.
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.
脊椎动物骨骼肌的α-原肌球蛋白以头对头的方式聚合，并与肌动蛋白协同结合。据推测，肌动蛋白的协同结合受头对头相互作用强度的支配。为了了解头对头亲和力与肌动蛋白结合之间的关系，我们研究了在丝氨酸-283处有单个残基取代的原肌球蛋白变体的特性，丝氨酸-283是羧基末端的倒数第二个残基，参与头对头相互作用。研究表明，丝氨酸-283的磷酸化增强了头对头相互作用。采用粘度测定法比较原肌球蛋白变体的头对头亲和力。与野生型未磷酸化的α-原肌球蛋白相比，变体S283E的粘度更高，而变体S283K的粘度更低。结果证实了这种相互作用对残基283的离子特性敏感的观点。使用两对设计成仅允许二聚体相互作用的原肌球蛋白变体，通过沉降平衡直接评估头对头相互作用的强度。对于一对含有丝氨酸-283的变体，缔合常数测定为2.6×10⁴ M⁻¹（标准差 = 1.0×10⁴），而对于另一对含有谷氨酸-283的变体，亲和力为3.9×10⁴ M⁻¹（标准差 = 1.6×10⁴），略强于前者，与粘度测定结果一致。结果表明，头对头缔合如先前从光散射测量中所暗示的那样较弱。采用共沉降法测量原肌球蛋白变体与肌动蛋白的协同结合。尽管先前的结果表明磷酸化对肌动蛋白亲和力没有显著影响，但与对照相比，变体S283E的亲和力较低。变体S283K和S283A与肌动蛋白的亲和力甚至更低，尽管这些变体与肌动蛋白的结合比变体S283E更具协同性。结果表明，相邻原肌球蛋白分子之间头对头相互作用的亲和力较弱，并且在很大程度上受残基283处额外电荷的影响。另一方面，与肌动蛋白的相互作用、肌动蛋白结合中的亲和力和协同性取决于283位的氨基酸残基，并且与溶液中原肌球蛋白分子之间头对头相互作用的强度并非简单相关。
