Structural changes of fibronectin adsorbed to model surfaces probed by fluorescence resonance energy transfer
Structural changes of fibronectin adsorbed to model surfaces probed by fluorescence resonance energy transfer
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DOI：
10.1002/jbm.a.30026
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发表时间：
2004-06-01
影响因子：
4.9
通讯作者：
Vogel, V
中科院分区：
文献类型：
--
作者：
Baugh, L;Vogel, V
蛋白质在生物材料吸附过程中的结构变化会影响分子结合部位的呈现，最终影响生物材料的性能。我们应用荧光共振能量转移(FRET)光谱研究了细胞黏附蛋白纤维连接蛋白(FN)在模拟亲水和疏水表面吸附后的结构变化。使用两种标记方案用供体和受体荧光团标记FN，并根据测量到的FN在变性溶液中的结构变化来校准分子内的能量转移。然后用FRET测量了FN的表面结构。在FRET的基础上，FN在亲水性玻璃上的二聚体臂比在疏水性氟烷基硅烷衍生玻璃(氟硅烷)上的伸展程度更大，并且在一定的吸附浓度范围内这种伸展对分子堆积不敏感。FN在玻璃上的构象比在氟硅烷上更能促进细胞附着；讨论了整体结构变化(模块移动)和局部结构变化(二级结构破坏)对FN细胞整合素结合活性的影响。在FRET工作的基础上，我们将FN在这些表面上的构象与其在成纤维细胞培养中的构象进行了比较。FRET的独特之处在于可以直接比较生物材料表面和细胞培养之间的蛋白质结构。(C)2004年威利期刊公司。
Structural changes of proteins during adsorption to biomaterials affect the presentation of molecular binding sites and, ultimately, biomaterial performance. We have applied fluorescence resonance energy transfer (FRET) spectroscopy to study structural changes of the cell adhesion protein, fibronectin (Fn), following adsorption to model hydrophilic and hydrophobic surfaces. Fn was labeled with donor and acceptor fluorophores using two labeling schemes and intramolecular energy transfer was calibrated against measured structural changes of Fn in denaturing solutions. FRET was then applied to measure Fn's structure on surfaces. Based on FRET, Fn underwent greater extension of its dimer arms on hydrophilic glass than on hydrophobic fluoroalklysilane-derivatized glass (fluorosilane), and this extension was insensitive to molecular packing over a range of adsorption concentrations. Fn's conformation on glass better promoted cell attachment than on fluorosilane; the roles of both global structural changes (movements of modules) and local structural changes (disruption of secondary structure) on Fn's cell integrin binding activity are discussed. Based on previous FRET work, we compare Fn's conformations on these surfaces with its conformations in fibroblast culture. FRET is unique in allowing direct comparison of protein structure between biomaterial surfaces and cell culture. (C) 2004 Wiley Periodicals, Inc.