Patel Salima, Chaffotte Alain F, Amana Batt, Goubard Fabrice, Pauthe Emmanuel
Department of Physiology and Biophysics, ERRMECe, Université de Cergy-Pontoise, 95302 Cergy-Pontoise, Cedex, France.
Int J Biochem Cell Biol. 2006;38(9):1547-60. doi: 10.1016/j.biocel.2006.03.005. Epub 2006 Mar 28.
It is well established that fibronectin into extracellular matrix undergoes repeated tensions applied by cells, resulting into dramatic structural changes which reflect its elastic properties. However, there is currently no study reporting with precision the consequences of this elasticity on fibronectin structure and conformation. In the present work, we investigated fibronectin structural and conformational reorganization in vitro through a denaturation-renaturation approach. The similarities and differences between "refolded fibronectin" and "native fibronectin" were investigated using various spectroscopic methods, hydrodynamic characterization, molecular imaging and biochemical characterization. In the refolded form, secondary structure elements as well as local tyrosine and tryptophan environment are identical compared to the native form. Interestingly, some differences in global tertiary structure organization and molecular conformation were observed. These differences are due to the reactivity of the two free cysteines, which are buried in the native state but become accessible during the unfolding process. First, oxidation of these residues leading to the formation of intermolecular disulfide bonds results in formation of stabilized multimer. Second, some illegitimate intramolecular disulfide bonds are formed. The presence of iodoacetamide, the sulfhydryl alkylating agent, during the unfolding-refolding process prevents all these events. This study clearly demonstrates that, under near physiological conditions, competitive renaturation pathways occur, involving free cysteines in either multimer formation or intermolecular shuffling of disulfide bonds. These findings might have important implications for future studies and be helpful to develop a deeper understanding of fibronectin morphology.
众所周知，细胞外基质中的纤连蛋白会受到细胞施加的反复张力，从而导致显著的结构变化，这反映了其弹性特性。然而，目前尚无研究精确报道这种弹性对纤连蛋白结构和构象的影响。在本研究中，我们通过变性-复性方法在体外研究了纤连蛋白的结构和构象重组。使用各种光谱方法、流体动力学表征、分子成像和生化表征研究了“重折叠纤连蛋白”和“天然纤连蛋白”之间的异同。在重折叠形式中，二级结构元件以及局部酪氨酸和色氨酸环境与天然形式相同。有趣的是，观察到全局三级结构组织和分子构象存在一些差异。这些差异是由于两个游离半胱氨酸的反应性，它们在天然状态下被掩埋，但在展开过程中变得可及。首先，这些残基的氧化导致分子间二硫键的形成，从而形成稳定的多聚体。其次，形成了一些不合法的分子内二硫键。在展开-重折叠过程中存在碘乙酰胺（巯基烷基化剂）可阻止所有这些事件。本研究清楚地表明，在接近生理条件下，会发生竞争性的复性途径，涉及游离半胱氨酸参与多聚体形成或二硫键的分子间重排。这些发现可能对未来的研究具有重要意义，并有助于更深入地理解纤连蛋白的形态。
