Jancsó A, Graceffa P
Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
J Biol Chem. 1991 Mar 25;266(9):5891-7.
Subunits of gizzard smooth muscle tropomyosin, dissociated by guanidinium chloride and reassociated by high salt dialysis, form a 1:1 mixture of the beta beta and gamma gamma homodimers (Graceffa, P. (1989) Biochemistry 28, 1282-1287). The homodimers have now been separated by anion-exchange chromatography and native gel electrophoresis, enabling us to show that the native protein is composed of more than 90% heterodimer. The in vitro equilibrium distribution of heterodimer and homodimers, at close to physiological temperature and ionic conditions, was calculated from thermal unfolding profiles of separated homodimers and heterodimer, as monitored by circular dichroism. The results, for an equal proportion of beta and gamma chains, indicate a predominant formation of heterodimer via chain dissociation and chain exchange, although the proportion of heterodimer was much less than the 90-100% found in the native protein. However, the proportion of heterodimer for actin-bound tropomyosin, determined by analyzing tropomyosin sedimented with actin, was greater than 90%, which may provide a model for assembly in vivo. The end-to-end interactions of the homodimers are about the same but are much less than that of the native heterodimer, as determined by viscometry. The greater end-to-end interaction of heterodimers may lead to stronger binding to actin compared to homodimers and thus would further shift the equilibrium between heterodimer and homodimers toward heterodimer and possibly account for the almost exclusive population of heterodimer in the presence of actin. The greater end-to-end interaction of the heterodimer may also provide a functional advantage for its preferred assembly. This study also shows that the two-step thermal unfolding of the homodimer mixture is due to the formation of heterodimer via an intermediate which is a new type of tropomyosin species which forms a gel in low salt. This tropomyosin is also present in small amounts in native tropomyosin preparations.
砂囊平滑肌原肌球蛋白的亚基，经氯化胍解离并通过高盐透析重新缔合后，形成ββ和γγ同型二聚体的1:1混合物（格拉切法，P.（1989年）《生物化学》28卷，1282 - 1287页）。现在通过阴离子交换色谱法和天然凝胶电泳分离了这些同型二聚体，这使我们能够证明天然蛋白质由超过90%的异源二聚体组成。通过圆二色性监测，根据分离出的同型二聚体和异源二聚体的热解折叠曲线，计算了在接近生理温度和离子条件下异源二聚体和同型二聚体的体外平衡分布。对于等量的β链和γ链，结果表明通过链解离和链交换主要形成异源二聚体，尽管异源二聚体的比例远低于天然蛋白质中发现的90 - 100%。然而，通过分析与肌动蛋白一起沉淀的原肌球蛋白来确定，与肌动蛋白结合的原肌球蛋白中异源二聚体的比例大于90%，这可能为体内组装提供一个模型。通过粘度测定法确定，同型二聚体的端到端相互作用大致相同，但远小于天然异源二聚体。与同型二聚体相比，异源二聚体更大的端到端相互作用可能导致其与肌动蛋白的结合更强，从而进一步使异源二聚体和同型二聚体之间的平衡向异源二聚体方向移动，并可能解释在有肌动蛋白存在时异源二聚体几乎占主导的情况。异源二聚体更大的端到端相互作用也可能为其优先组装提供功能优势。这项研究还表明，同型二聚体混合物的两步热解折叠是由于通过一种中间体形成异源二聚体，该中间体是一种新型的原肌球蛋白物种，在低盐条件下形成凝胶。这种原肌球蛋白在天然原肌球蛋白制剂中也少量存在。
