Fluorescence energy transfer between the myosin subfragment-1 isoenzymes and F-actin in the absence and presence of nucleotides.
Fluorescence energy transfer between the myosin subfragment-1 isoenzymes and F-actin in the absence and presence of nucleotides.
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在存在和不存在核苷酸的情况下，肌球蛋白亚片段 1 同工酶和 F-肌动蛋白之间的荧光能量转移。
DOI：
10.1111/j.1432-1033.1983.tb07616.x
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
I. Trayer
中科院分区：
文献类型：
--
作者：
H. Trayer;I. Trayer
通过胰凝乳蛋白酶消化制备的肌球蛋白亚片段 1 (S1) 的独特快速反应半胱氨酸残基 (SH1) 和肌动蛋白的半胱氨酸 373 已用荧光探针 N-(溴乙酰基)-N'-(1-磺基-5-萘基)乙二胺 (1,5-BrAEDANS) 和 5-(碘乙酰胺基)荧光素选择性标记（5-IAF），其光谱特性使其成为荧光能量转移研究中特别有效的供体-受体对。 40-45% 的转移效率代表约 5 nm 的发色团空间分离，这与之前报道的通过木瓜蛋白酶消化制备的类似标记的 S1 和肌动蛋白的 6 nm 值相当一致 [Takashi, R. (1979) Biochemistry, 18, 5164-5169]。当形成双标记二元复合物时，这种转移效率没有变化：（1）使用acto-S1（A1）或acto-S1（A2）在10-200 mM KCl，pH 7-8和不同的缓冲条件下； (2) 在存在和不存在 Ca2+ 的情况下或当供体和受体附着位点颠倒时，使用 S1 同工酶和受调节的肌动蛋白（即肌动蛋白与原肌球蛋白和肌钙蛋白）。对供体和受体偏振荧光的分析表明，发色团不是随机定向的（即 chi 2 不等于 2/3），但它们确实相对于任一蛋白质有一些运动。根据对 chi 2 极限值的了解，供体和受体发色团的位点间距离计算为在 3.9-6.7 nm 范围内。将 MgATP 添加到双标记 acto-S1 复合物中消除了能量转移，但当 ATP 水解完成时能量转移又恢复了。利用 S1、肌动蛋白和 MgADP 或 MgAdoPP[NH]P（腺苷镁 5'-β，γ-亚氨基]三磷酸）之间的已知结合常数 [Konrad, M. 和 Goody, K. (1982) Eur. J.生物化学。 128、547-555； Greene, L. E. 和 Eisenberg, E. (1980) J. Biol。化学。 255, 543-548]，测定了平衡时存在的所有物质的浓度。选择实验条件以最大化三元acto-S1-核苷酸复合物的量（约等于50%）并最小化二元复合物的量（小于或等于2%）。发现三元复合物中发色团相互作用位点的空间分离与两种核苷酸相同，并且与二元复合物中发现的没有区别。采用类似的策略通过 1 H-NMR 光谱比较二元和三元配合物的构象。在这些实验中，大约 90% 的 S1 是三元配合物的形式。添加 MgAdoPP[NH]P 或 MgADP 后，acto-S1 光谱没有明显变化。这些观察结果支持这样的结论：当“严格的”二元 acto-S1 复合物结合 ADP 或 AdoPP[NH]P 时，其结构没有大的变化。
The unique fast-reacting cysteine residue (SH1) of myosin subfragment 1 (S1), prepared by chymotryptic digestion, and cysteine 373 of actin have been labelled selectively with the fluorescent probes, N-(bromoacetyl)-N'-(1-sulpho-5-naphthyl)ethylenediamine (1,5-BrAEDANS) and 5-(iodoacetamido)fluorescein (5-IAF), whose spectral properties render them a particularly effective donor-acceptor pair in fluorescence energy transfer studies. The transfer efficiency of 40-45% represented a spatial separation of the chromophores of about 5 nm, which is in reasonable agreement with the value of 6 nm reported earlier for similarly labelled S1, prepared by papain digestion, and actin [Takashi, R. (1979) Biochemistry, 18, 5164-5169]. This transfer efficiency did not change when the doubly-labelled binary complex was formed: (1) with acto-S1(A1) or acto-S1(A2) at 10-200 mM KCl, pH 7-8 and different buffer conditions; (2) with either S1 isoenzyme and regulated actin (i.e. actin with tropomyosin and troponin) both in the presence and absence of Ca2+ or when the donor and acceptor attachment sites were reversed. Analysis of donor and acceptor polarized fluorescence showed that the chromophores are not randomly orientated (i.e. chi 2 not equal to 2/3), but they do have some motion relative to either protein. From a knowledge of the limiting values for chi 2, the intersite distance for donor and acceptor chromophores was calculated to be in the range 3.9-6.7 nm. Addition of MgATP to the doubly-labelled acto-S1 complex eliminated energy transfer but this was recovered when ATP hydrolysis was completed. By utilizing the known binding constants between S1, actin and either MgADP or MgAdoPP[NH]P (magnesium adenosine 5'-[beta, gamma-imido]triphosphate) [Konrad, M. and Goody, K. (1982) Eur. J. Biochem. 128, 547-555; Greene, L. E. and Eisenberg, E. (1980) J. Biol. Chem. 255, 543-548], the concentrations of all species present at equilibrium were determined. Experimental conditions were chosen to maximise the amount of ternary acto-S1-nucleotide complex (approximately equal to 50%) and minimise the amount of binary complex (less than or equal to 2%). The spatial separation of the chromophore interaction sites in the ternary complex was found to be the same with both nucleotides and indistinguishable from that found with the binary complex. A similar strategy was employed to compare the conformations of the binary and ternary complexes by 1H-NMR spectroscopy. In these experiments about 90% of the S1 was in the form of the ternary complex. There was no noticeable change in the acto-S1 spectra upon addition of either MgAdoPP[NH]P or MgADP. These observations support the conclusion that there is no large change in structure in the 'rigor' binary acto-S1 complex when it binds either ADP or AdoPP[NH]P.
影响因子：
5.6
作者：
Silberstein,L;Lowey,S
通讯作者：
Lowey,S
肌球蛋白亚片段 1 的快速反应硫醇和反应性赖氨酸残基之间的空间关系。
影响因子：
2.9
作者：
Takashi,R;Muhlrad,A;Botts,J
通讯作者：
Botts,J
腺苷 5-二磷酸和腺苷 5-（β，γ-亚胺三磷酸）与亚片段 1 和 acto 亚片段 1 结合的瞬时动力学。
影响因子：
2.9
作者：
Trybus,KM;Taylor,EW
通讯作者：
Taylor,EW