Center for Nanotechnology, Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
Colloids Surf B Biointerfaces. 2009 Dec 1;74(2):401-9. doi: 10.1016/j.colsurfb.2009.07.004. Epub 2009 Jul 14.
The growth of natural biominerals is often tightly regulated by surface adsorption and subsequent incorporation of proteins into the crystal structure. Understanding how macromolecules intercalate into inorganic crystal lattices and how incorporation affects protein structure is crucial to learning how to engineer biomimetic materials with advanced properties, yet knowledge about the molecular-level interactions between organic guests and inorganic hosts remains sparse. Here we have used fluorescence resonance energy transfer (FRET) to probe conformational changes of a macromolecule as it adsorbs to, and becomes incorporated within, a biomineral crystal. Calcium oxalate monohydrate (COM) was used as a model due to its large size and kinetic stability under a wide range of pH values. Since the conformation of the extracellular matrix protein fibronectin (Fn) is highly sensitive to local ion concentrations, major conformational changes can be observed by FRET, as Fn senses and responds to varying local ionic conditions. When transferred from a physiological buffer to a supersaturated solution, Fn's crossed-over dimeric arms separate, indicating a weakening of the electrostatic interactions which otherwise stabilize the compact conformation of the protein. Fn returns to a more compact state when binding to the flat (-101) surface of the crystal, suggesting that Fn might sense a zone of ion depletion right at the interface of the growing crystal. As the crystal begins to grow around the absorbed protein, the dimeric Fn arms separate again, potentially driven by interactions with the newly formed charged step edges forming around it during the embedding process. FRET thus reveals for the first time how local changes in the electrostatic environment during the growth of a biomineral can cause major alterations in protein conformation. The insights derived using FRET and atomic force microscopy (AFM) could stimulate novel ways to tailor and tune the properties of organic-inorganic composites by exploiting dynamically changing electrostatic guest-host interactions.
天然生物矿的生长通常受到表面吸附的严格调控，随后蛋白质被整合到晶体结构中。了解大分子如何插入无机晶格以及整合如何影响蛋白质结构对于学习如何用具有先进性能的仿生材料进行工程设计至关重要，但关于有机客体和无机主体之间的分子水平相互作用的知识仍然很少。在这里，我们使用荧光共振能量转移（FRET）来探测大分子在吸附到生物矿晶体并被整合到其中时的构象变化。草酸钙一水合物（COM）被用作模型，因为它的尺寸大，在广泛的 pH 值范围内具有动力学稳定性。由于细胞外基质蛋白纤维连接蛋白（Fn）的构象对局部离子浓度高度敏感，因此可以通过 FRET 观察到主要的构象变化，因为 Fn 可以感知并响应不断变化的局部离子条件。当从生理缓冲液转移到过饱和溶液时，Fn 的交叉二聚臂分离，表明静电相互作用减弱，否则会稳定蛋白质的紧凑构象。当 Fn 结合到晶体的平坦（-101）表面时，Fn 会回到更紧凑的状态，这表明 Fn 可能会感觉到在生长晶体的界面处存在离子耗尽区。当晶体开始在被吸收的蛋白质周围生长时，二聚 Fn 臂再次分离，这可能是由与在嵌入过程中围绕其形成的新形成的带电阶跃边缘的相互作用驱动的。FRET 首次揭示了在生物矿生长过程中局部静电环境的变化如何导致蛋白质构象的重大改变。使用 FRET 和原子力显微镜（AFM）获得的见解可以通过利用动态变化的静电客体-主体相互作用来刺激定制和调整有机-无机复合材料性能的新方法。
