Singh Kulwant, Bhakuni Vinod
Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India.
J Biochem. 2009 Jun;145(6):721-31. doi: 10.1093/jb/mvp029. Epub 2009 Feb 23.
Usually during the folding/unfolding of flavoproteins, an apo-intermediate is stabilized before global unfolding of the enzymes occurs. However, stabilization of a holo-intermediate has also been reported for a few flavoproteins. We have studied the unfolding of Toxoplasma gondii ferredoxin-NADP+ reductase (TgFNR) using GdnHCl and urea. A functionally inactive holo-intermediate of the enzyme was found to be stabilized during this unfolding process. The intermediate species had cofactor FAD bound to it, but it showed free movement due to which the stabilized intermediates were functionally inactive. The native TgFNR behaves cooperatively with the two structural domains interacting strongly with each other. The denaturants GdnHCl and urea, at low concentrations, were found to interact selectively with the NADP+-binding domain of TgFNR and to induce structural modifications in it. These selective modifications in the protein molecule lead to loss of interactions between two domains and the enzyme behaved non-cooperatively resulting in stabilization of an intermediate species. Significant differences in the structural properties of the GdnHCl- and urea-stabilized holo-intermediates of TgFNR were observed. Comparison of the unfolding pathway of TgFNR (a plant-type FNR) with that of FprA (a GR-type FNR) demonstrates that they follow very different pathways of unfolding.
通常在黄素蛋白折叠/去折叠过程中，脱辅基中间体在酶整体去折叠之前就已稳定。然而，也有报道称少数黄素蛋白存在全酶中间体的稳定化现象。我们使用盐酸胍（GdnHCl）和尿素研究了刚地弓形虫铁氧化还原蛋白-NADP⁺还原酶（TgFNR）的去折叠过程。发现该酶的一种功能无活性的全酶中间体在这一去折叠过程中得以稳定。该中间物种结合有辅因子FAD，但由于其显示出自由移动，所以稳定化的中间体在功能上无活性。天然的TgFNR表现出协同性，其两个结构域之间相互强烈作用。发现低浓度的变性剂盐酸胍和尿素会选择性地与TgFNR的NADP⁺结合结构域相互作用，并在其中诱导结构修饰。蛋白质分子中的这些选择性修饰导致两个结构域之间的相互作用丧失，酶表现出非协同性，从而使一种中间物种得以稳定。观察到TgFNR的盐酸胍和尿素稳定化全酶中间体在结构性质上存在显著差异。将TgFNR（一种植物型FNR）的去折叠途径与FprA（一种GR型FNR）的去折叠途径进行比较表明，它们遵循非常不同的去折叠途径。
