Miki M
Department of Anatomy, University of Sydney, Australia.
Eur J Biochem. 1990 Jan 12;187(1):155-62. doi: 10.1111/j.1432-1033.1990.tb15289.x.
The spatial relationships between Lys-61, Cys-374 on actin or SH1 on myosin subfragment-1 (S1) and Cys-190 on tropomyosin or Cys-133 on troponin-I (TnI) in a reconstituted thin filament were studied by fluorescence resonance energy transfer. 5-(2-Iodoacetylaminoethyl)aminonaphthalene 1-sulfonic acid (IAEDANS) attached to Lys-190 on tropomyosin or to Cys-133 on TnI was used as a donor. Fluorescein 5-isothiocyanate (FITC) attached to Lys-61 or 5-(iodoacetoamido)fluorescein (IAF) attached to Cys-374 on actin and 4-dimethylaminophenyl-azophenyl 4'-maleimide (DABMI) attached to SH1 on S1 were used as an acceptor. The transfer efficiency between AEDANS attached to Cys-190 on tropomyosin and FITC attached to Lys-61 on actin was 0.42 in the absence of troponin, 0.46 in the presence of troponin and Ca2+ and 0.55 in the presence of troponin and absence of Ca2+. The corresponding distances between the probes were calculated to be 4.7 nm, 4.6 nm and 4.3 nm respectively, assuming a random orientation factor K2 = 2/3. A large difference in the transfer efficiency from AEDANS attached to Cys-133 on TnI to FITC attached to Lys-61 on actin was observed between in the presence (0.52) and absence (0.70) of Ca2+. The corresponding distances between the probes were calculated to be 4.5 nm in the presence of Ca2+ and 3.9 nm in the absence of Ca2+. The distance between Cys-190 on tropomyosin and Cys-374 on actin was measured to be 5.1 nm and the transfer efficiency (0.35) did not change upon addition of troponin whether Ca2+ is present or not, in agreement with the previous report [Tao, T., Lamkin, M. & Lehrer, S. S. (1983) Biochemistry 22, 3059-3064]. The distance between Cys-133 on TnI and Cys-374 on actin was measured to be 4.4 nm. No detectable change in transfer efficiency (0.58) was observed between values in the presence and absence of Ca2+. These results suggest that a relative movement of the two domains of actin monomer in a reconstituted thin filament occurs in response to a change in Ca2+ concentration. The transfer efficiencies between DABMI attached to SH1 on S1 and AEDANS attached to Cys-190 on tropomyosin or Cys-133 on TnI were too small (less than 2%) for an accurate estimation of the distances, suggesting the distances are longer than 7.3 nm.
通过荧光共振能量转移研究了重组细肌丝中肌动蛋白上的赖氨酸-61、半胱氨酸-374或肌球蛋白亚片段-1（S1）上的SH1与原肌球蛋白上的半胱氨酸-190或肌钙蛋白I（TnI）上的半胱氨酸-133之间的空间关系。连接到原肌球蛋白上的赖氨酸-190或TnI上的半胱氨酸-133的5-（2-碘乙酰氨基乙基）氨基萘-1-磺酸（IAEDANS）用作供体。连接到肌动蛋白上的赖氨酸-61的异硫氰酸荧光素（FITC）、连接到肌动蛋白上半胱氨酸-374的5-（碘乙酰氨基）荧光素（IAF）以及连接到S1上SH1的4-二甲基氨基苯基-偶氮苯基-4'-马来酰亚胺（DABMI）用作受体。在不存在肌钙蛋白的情况下，连接到原肌球蛋白上半胱氨酸-190的AEDANS与连接到肌动蛋白上赖氨酸-61的FITC之间的转移效率为0.42，在存在肌钙蛋白和Ca2+的情况下为0.46，在存在肌钙蛋白且不存在Ca2+的情况下为0.55。假设随机取向因子K2 = 2/3，探针之间的相应距离分别计算为4.7 nm、4.6 nm和4.3 nm。在存在Ca2+（0.52）和不存在Ca2+（0.70）的情况下，观察到连接到TnI上半胱氨酸-133的AEDANS与连接到肌动蛋白上赖氨酸-61的FITC之间的转移效率有很大差异。探针之间的相应距离在存在Ca2+时计算为4.5 nm，在不存在Ca2+时计算为3.9 nm。测量到原肌球蛋白上的半胱氨酸-190与肌动蛋白上的半胱氨酸-374之间的距离为5.1 nm，并且无论是否存在Ca2+，添加肌钙蛋白后转移效率（0.35）都没有变化，这与先前的报告一致[陶，T.，拉姆金，M. & 莱勒，S. S.（1983年）《生物化学》22，3059 - 3064]。测量到TnI上的半胱氨酸-133与肌动蛋白上的半胱氨酸-374之间的距离为4.4 nm。在存在和不存在Ca2+的情况下，转移效率（0.58）没有观察到可检测到的变化。这些结果表明，重组细肌丝中肌动蛋白单体的两个结构域会响应Ca2+浓度的变化而发生相对移动。连接到S1上SH1的DABMI与连接到原肌球蛋白上半胱氨酸-190或TnI上半胱氨酸-133的AEDANS之间的转移效率太小（小于2%），无法准确估计距离，表明距离大于7.3 nm。
