Effects on the conformation of FVIIa by sTF and Ca2+ binding: Studies of fluorescence resonance energy transfer and quenching
Effects on the conformation of FVIIa by sTF and Ca2+ binding: Studies of fluorescence resonance energy transfer and quenching
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DOI：
10.1016/j.bbrc.2011.08.135
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发表时间：
2011-10-07
影响因子：
3.1
通讯作者：
Svensson, Magdalena
中科院分区：
文献类型：
--
作者：
Carlsson, Karin;Persson, Egon;Svensson, Magdalena
通过 FRET 分析估计溶液中 FVIIa 的表观长度。两种荧光探针，荧光素（Fl-FPR）和罗丹明衍生物（TMR），共价连接至FVIIa。 Fl-FPR 的结合位点位于蛋白酶结构域中，而 TMR 位于 Gla 结构域中，因此允许对蛋白质的几乎整个延伸进行长度测量。根据 FRET 测量，两个探针之间的距离对于游离 FVIIa 确定为 61.4 埃，对于与可溶性组织因子 (sTF) 结合的 FVIIa 确定为 65.5 埃。与基于复杂晶体结构预期的距离相比，这些看似很短的距离需要探针相互拉伸。因此，在溶液中与 sTF 形成复合物后，FRET 分析的表观距离增加了 4 埃。然而，考虑到基于 FVIIa 和 sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044-1053) 的最新分子动力学模拟的蛋白质动力学如何影响表观荧光信号，我们的计算表明，活性位点抑制的 FVIIa 的整体平均构象在连接至sTF。从酰胺分解活性测量可知，Ca2+结合会导致FVIIa的激活，但我们首次直接证明了Ca2+结合后活性位点环境的构象变化。有趣的是，即使在存在抑制剂的情况下，也可以注意到这种 Ca2+ 诱导的构象变化。与 sTF 形成复合物进一步稳定了这种构象变化，从而形成更难以接近的活性位点探针。 (C) 2011 Elsevier Inc. 保留所有权利。
The apparent length of FVIIa in solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein (Fl-FPR) and a rhodamine derivative (TMR), were covalently attached to FVIIa. The binding site of Fl-FPR was in the protease domain whereas TMR was positioned in the Gla domain, thus allowing a length measure over virtually the whole extension of the protein. From the FRET measurements, the distances between the two probes were determined to be 61.4 for free FVIIa and 65.5 angstrom for FVIIa bound to soluble tissue factor (sTF). These seemingly short distances, compared to those anticipated based on the complex crystal structure, require that the probes stretch towards each other. Thus, the apparent distance from the FRET analysis was shown to increase with 4 angstrom upon formation of a complex with sTF in solution. However, considering how protein dynamics, based on recent molecular dynamics simulations of FVIIa and sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044-1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF.It is known from amidolytic activity measurements that Ca2+ binding leads to activation of FVIIa, but we have for the first time directly demonstrated conformational changes in the environment of the active site upon Ca2+ binding. Interestingly, this Ca2+-induced conformational change can be noted even in the presence of an inhibitor. Forming a complex with sTF further stabilized this conformational change, leading to a more inaccessible active-site located probe. (C) 2011 Elsevier Inc. All rights reserved.