Goder V, Beckert V, Pfeil W, Bernhardt R
Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Strasse 10, Berlin-Buch, D-13125, Germany.
Arch Biochem Biophys. 1998 Nov 1;359(1):31-41. doi: 10.1006/abbi.1998.0873.
Bovine preadrenodoxin, an adrenocortical precursor protein destined for mitochondrial import, was expressed in Escherichia coli as an [2Fe-2S] cluster-containing protein. It was found in inclusion bodies, purified from there, and finally reconstituted to obtain soluble holo-protein. The impact of the presequence on folding of the protein using biochemical and biophysical approaches has been investigated. Upon unfolding the preprotein reveals a decrease in the denaturational enthalpy and heat capacity compared with mature adrenodoxin, indicating an incomplete unfolding of the preprotein with remaining residual structure. Moreover, the data obtained show that the presequence is solvent exposed in aqueous solution with no preference for secondary structure elements and that it does not disturb the accurate folding of the mature part of the protein. The latter conclusion is also based on the finding that the precursor in vitro exhibits electron transfer function comparable to the mature protein, adrenodoxin. While the reduction of cytochrome c, reflecting the interaction between adrenodoxin and its reductase, and the interaction with CYP11B1 have not been significantly affected by the presence of the presequence, the binding affinity of preadrenodoxin to CYP11A1 is 5.5-fold lower than that of the mature form.
牛前肾上腺皮质铁氧还蛋白是一种注定要导入线粒体的肾上腺皮质前体蛋白，在大肠杆菌中表达为一种含[2Fe-2S]簇的蛋白。它存在于包涵体中，从那里纯化出来，最后进行重构以获得可溶性全蛋白。已使用生化和生物物理方法研究了前导序列对该蛋白折叠的影响。与成熟的肾上腺皮质铁氧还蛋白相比，前体蛋白在展开时变性焓和热容降低，表明前体蛋白展开不完全，仍有残余结构。此外，获得的数据表明，前导序列在水溶液中暴露于溶剂中，对二级结构元件没有偏好，并且它不会干扰该蛋白成熟部分的准确折叠。后一个结论还基于以下发现：该前体在体外表现出与成熟蛋白肾上腺皮质铁氧还蛋白相当的电子传递功能。虽然反映肾上腺皮质铁氧还蛋白与其还原酶之间相互作用的细胞色素c还原以及与CYP11B1的相互作用并未因前导序列的存在而受到显著影响，但前肾上腺皮质铁氧还蛋白与CYP11A1的结合亲和力比成熟形式低5.5倍。
