Fluorescence energy transfer detects changes in fibronectin structure upon surface binding.
Fluorescence energy transfer detects changes in fibronectin structure upon surface binding.
复制标题
荧光能量转移检测表面结合时纤连蛋白结构的变化。
DOI：
10.1016/0003-9861(89)90320-2
复制
发表时间：
1989
影响因子：
3.9
通讯作者：
Lai,CS
中科院分区：
文献类型：
--
作者：
Wolff,C;Lai,CS
在此，我们报告了利用荧光能量转移技术检测到的人血浆纤维连接蛋白（Fn）分子内距离的变化，该蛋白被吸附到Cytodex葡聚糖微载体表面。在每条链的氨基末端附近的谷氨酰胺-3残基，使用凝血因子XIIIa，用单聚氨嘧啶尸胺（dansyl）或单荧光素尸胺（fluorescein）进行酶标记。利用这个供体（丹酰）-受体（荧光素）对和稳态测量，我们先前证明了溶液中血浆纤维连接蛋白的两个氨基端是并列的，并被23 Å (C. Wolff和C. s。赖（1988）生物化学27,3483-3487)。在微载体上吸附后，发现能量转移完全消除，表明表面结合引起构象变化，使两个氨基端之间的距离增加到70以上Å。此外，我们用荧光素标记了每条链的氨基末端，用香豆素基苯基马来酰亚胺标记了每条链的两个游离巯基，香豆素基苯基马来酰亚胺作为能量供体。双标记蛋白在溶液中的发射光谱显示发生了能量转移，表明氨基端与游离巯基(s)之间的相对距离在70以内Å。在表面结合时，也注意到供体-受体对之间能量转移的减少。这里提出的结果与等离子体Fn在表面结合时经历剧烈构象变化的观点一致，可能从紧凑形式转变为扩展形式。这一过程可能对纤连蛋白分子的表面活化很重要。
We report here the changes in intramolecular distances in human plasma fibronectin (Fn) detected, upon adsorption of the protein to the surface of the Cytodex dextran microcarrier, using a fluorescence energy transfer technique. The glutamine-3 residue, near the amino terminus of each chain, was labeled enzymatically with either monodansylcadaverine (dansyl) or monofluoresceinylcadaverine (fluorescein) by use of coagulation factor XIIIa. Using this donor (dansyl)-acceptor (fluorescein) pair, and steady-state measurements, we demonstrated previously that the two amino termini of plasma fibronectin in solution were juxtaposed and separated by 23 Å (C. Wolff and C.-S. Lai (1988)Biochemistry27, 3483–3487). Upon adsorption to the microcarrier, the energy transfer was found to be completely abolished, suggesting that the surface binding induces a conformational change by which the distance between the two amino termini is increased to more than 70 Å. Moreover, we have labeled the amino terminus of each chain with fluorescein and the two free sulfhydryl groups of each chain with coumarinyl-phenylmaleimide which serves as an energy donor. The emission spectra of the double-labeled protein in solution showed the occurrence of energy transfer, indicating that the relative distances between the amino termini and the free sulfhydryl group(s) are within 70 Å. Upon surface binding, a decrease in the energy transfer between this donor-acceptor pair was also noted. The results presented here are consistent with the notion that plasma Fn undergoes a drastic conformational change upon surface binding, perhaps changing from a compact form to an extended form. This process may be important for the surface activation of the fibronectin molecule.