Binding and release of iron by gel-encapsulated human transferrin: evidence for a conformational search.
Binding and release of iron by gel-encapsulated human transferrin: evidence for a conformational search.
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凝胶封装的人转铁蛋白结合和释放铁：构象搜索的证据。
DOI：
10.1073/pnas.262526399
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Friedman,JoelM
中科院分区：
文献类型：
--
作者：
Navati,MahanteshS;Samuni,Uri;Aisen,Philip;Friedman,JoelM
人转铁蛋白是一种单链双叶蛋白，两个相似但不相同的叶依次由两个结构域组成。每个叶瓣可以采用两种稳定的结构构象之一，开放或封闭，由畴相对于彼此的刚性旋转决定。在溶液中，开放和封闭构象之间的叶瓣转换与Fe（III）离子的释放或结合有关。本研究的结果表明，将转铁蛋白包封在多孔溶胶-凝胶基质中，可以将这种相互转化时间急剧延长至数天或数周，从而为探测迄今为止无法获得的瞬态中间体提供了机会。采用两种方法制备了无铁转铁蛋白溶胶-凝胶包封样品。在第一种方案中，载转铁蛋白的平衡形式被包裹在溶胶-凝胶基质中，而在第二种方案中，全转铁蛋白首先被包裹，然后从蛋白质中去除铁。动力学和光谱研究的结果允许区分铁结合的两种模型。首先，假设铁与一个结构域的氨基酸配体结合，诱导第二个结构域的刚性旋转，以关闭结构域间的间隙。在第二种情况下，铁在叶瓣的热可达状态中进行构象搜索，“选择”最接近铁被束缚时稳定闭合状态的状态。我们的实验结果支持第二种机制。
Human transferrin is a single-chain bilobal protein with each of the two similar but not identical lobes in turn composed of two domains. Each lobe may assume one of two stable structural conformations, open or closed, determined by a rigid rotation of the domains with respect to each other. In solution, the transformation of a lobe between open and closed conformations is associated with the release or binding of an Fe(III) ion. The results of the present study indicate that encapsulation of transferrin within a porous sol-gel matrix allows for a dramatic expansion, to days or weeks, of this interconversion time period, thus providing an opportunity to probe heretofore inaccessible transient intermediates. Sol-gel-encapsulated iron-free transferrin samples are prepared by using two protocols. In the first protocol, the equilibrium form of apotransferrin is encapsulated in the sol-gel matrix, whereas in the second protocol holotransferrin is first encapsulated and then iron is removed from the protein. Results of kinetic and spectroscopic studies allow for distinguishing between two models for iron binding. In the first, iron is assumed to bind to amino acid ligands of one domain, inducing a rigid rotation of the second domain to effect closure of the interdomain cleft. In the second, iron undertakes a conformational search among the thermally accessible states of the lobe, “choosing” the state which most nearly approximates the stable closed state when iron is bound. Our experimental results support the second mechanism.