Navati Mahantesh S, Samuni Uri, Aisen Philip, Friedman Joel M
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3832-7. doi: 10.1073/pnas.262526399. Epub 2002 Dec 16.
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.
人转铁蛋白是一种单链双叶蛋白，其两个相似但不完全相同的叶各自又由两个结构域组成。每个叶可呈现两种稳定的结构构象之一，即开放构象或闭合构象，这是由结构域之间相对于彼此的刚性旋转所决定的。在溶液中，叶在开放构象和闭合构象之间的转变与Fe(III)离子的释放或结合相关。本研究结果表明，将转铁蛋白包封在多孔溶胶 - 凝胶基质中可使这种相互转化的时间周期显著延长至数天或数周，从而提供了一个机会来探测迄今无法接近的瞬态中间体。通过两种方法制备溶胶 - 凝胶包封的无铁转铁蛋白样品。在第一种方法中，脱铁转铁蛋白的平衡形式被包封在溶胶 - 凝胶基质中，而在第二种方法中，首先包封全转铁蛋白，然后从蛋白质中去除铁。动力学和光谱学研究结果有助于区分两种铁结合模型。在第一种模型中，假定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实现结构域间裂隙的闭合。在第二种模型中，铁在叶的热可及状态之间进行构象搜索，“选择”当铁结合时最接近稳定闭合状态的状态。我们的实验结果支持第二种机制。
