Single-molecule determination of the face-specific adsorption of Amelogenin's C-terminus on hydroxyapatite.
Single-molecule determination of the face-specific adsorption of Amelogenin's C-terminus on hydroxyapatite.
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DOI：
10.1002/anie.201100181
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发表时间：
2011-08-08
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影响因子：
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通讯作者：
Wierzbicki A
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作者：
Friddle RW;Battle K;Trubetskoy V;Tao J;Salter EA;Moradian-Oldak J;De Yoreo JJ;Wierzbicki A
蛋白质-矿物质相互作用的能量学是矿化组织的分级结构的关键但表征不足的因素。在矿化过程中，有组织的蛋白质基质指导矿物质成分的形成。与所有组装过程一样，自由能的变化提供了潜在的热力学驱动力，在这种情况下，反映了蛋白质与新生mineral. However相互作用，尽管获得矿物结合的面特异性自由能的重要性，以建立分子水平的生物矿物组织的理解，迄今为止没有直接的测量报告。计算方法与蛋白质的复杂性、模型水电位的不足和背景电解质的影响作斗争。本文提出了一种新的力谱应用方法，即利用原子力显微镜（AFM）针尖，将Amelogenin蛋白（Amel）的C-末端片段功能化，直接测定Amel与牙釉质矿物相羟基磷灰石（HAp）结合的单分子面特异性自由能ΔGB。[1]然后，我们使用互补的分子动力学（MD）模拟比较结合能在不同的面和表面终端，并确定控制面特异性结合和晶体形态的关键相互作用。釉原蛋白（表1）是一种富含脯氨酸的疏水性蛋白质，自组装形成由大约100个单体组成的扁球形纳米颗粒[2]。[3，4]最近的体外结晶实验表明，Amel稳定磷酸钙（Ca-P）簇，其组装为复合Amel-Ca-P纳米簇。[5]这些簇共同组装成纳米颗粒链，这些纳米颗粒链演变成长的共同排列的晶体，类似于在生物釉质中发现的晶体。[5]晶体生长实验，[6，7] NMR光谱研究，[8-10]和中子散射[11]证明了Amel和HAp晶面之间的特定相互作用，其中Amel C-末端区域是必不可少的，后两种技术提供了C-末端足够接近HAp表面以指导生长的直接证据。牙釉质辅助的HAp形成导致具有限定牙釉质/唾液界面的基底（001）面的细长棱柱（100）晶体的表达。[12]这种晶体习性不同于未受抑制的无机生长的HAp，其表现出短的棱柱形（100）或平行四边形（001）形态，[13]或在骨形成期间生长的HAp晶体，其是沿c轴沿着伸长的薄板。[14因此，怀疑Amel结合到（100）面，从而抑制HAp沿沿着（100）生长并诱导c轴伸长。[16] LRAP是Amel的天然剪接变体，具有保留的N-和C-末端（表1），也是HAp生长的抑制剂，[17] LRAP的固态NMR光谱研究已经确定，LRAP C-末端区域的最后大约18个残基平坦地位于HAp表面上，具有显著的移动性，没有三维折叠。[8-10] LRAP在牙釉质发育中的作用尚未确定，但已提出促进牙釉质生长。[18]MD研究也提供了一些关于Amel-HAP结合几何结构的见解，[19]原则上可以描述结合的能量学。然而，虽然NMR数据提供了一个很好的实验约束的结构方面的模拟，没有这样的能量约束。基于生长抑制的测定是间接和定性的。定量探测相互作用自由能的实验方法通常基于所有面的平均体积测量。
The energetics of protein–mineral interactions is a crucial but poorly characterized factor underlying the hierarchical structure of mineralized tissue. During mineralization, organized protein matrices direct formation of mineral components. As with all assembly processes, the free-energy change provides the underlying thermodynamic driver, in this case reflecting protein interactions with the nascent mineral. However, despite the importance of obtaining face-specific free energies of mineral binding to establish a molecular-level understanding of biomineral organization, to date no direct measurements have been reported. Computational approaches struggle with the complexities of proteins, the inadequacies of model water potentials and effects of background electrolytes. Herein we present a novel application of force spectroscopy in which an atomic force microscopy (AFM) tip, functionalized with Amelogenin protein (Amel) C-terminal fragment, is used to directly determine the singlemolecule, face-specific free energy ΔGB of Amel binding to hydroxyapatite (HAp), the mineral phase in tooth enamel.[1] We then use complementary molecular dynamics (MD) simulations to compare binding energies at different faces and surface terminations and to identify the key interactions controlling face-specific binding and crystal morphology. Amelogenin (Table 1), a largely hydrophobic protein rich in proline, self-assembles to form oblate nanoparticles [2] comprised of approximately 100monomers.[3, 4] Recent invitro crystallization experiments show that Amel stabilizes calcium phosphate (Ca-P) clusters, which assemble as composite Amel-Ca-P nanoclusters.[5] These clusters co-assemble as chains of nanoparticles that evolve into long co-aligned crystals resembling those found in biological enamel.[5] Crystal-growth experiments,[6, 7] NMR spectroscopy studies,[8–10] and neutron scattering [11] demonstrate a specific interaction between Amel and HAp crystal faces, with the Amel C-terminal region implicated as essential, and with the latter two techniques providing direct evidence that the C-terminus is close enough to the HAp surface to direct growth. Amel-assisted HAp formation leads to expression of elongated prismatic (100) crystals with the basal (001) faces defining the enamel/saliva interface.[12] This crystal habit differs from that of uninhibited, inorganically grown HAp, which exhibits short, prismatic (100) or stubby (001) morphologies,[13] or HAp crystals grown during bone formation, which are thin plates elongated along the c-axis.[14, 15] Thus, it is suspected that Amel binds to the (100) face, thereby inhibiting HAp growth along (100) and inducing c-axis elongation.[16]Solid-state NMR spectroscopy studies of LRAP, which is both a naturally occurring splice variant of Amel with preserved N-and C-termini (Table1) and an inhibitor of HAp growth,[17] have established that the last approximately 18 residues of LRAP s C-terminal region lie flat on the HAp surface with significant mobility and without three-dimensional folding.[8–10] LRAP s role in enamel development has not been established, but promotion of enamel growth has been proposed.[18] MD studies, which have also provided some insights into the geometry of Amel-HAP binding,[19] can in principle delineate the energetics of binding. However, while NMR data provide a good experimental constraint on the structural aspects of simulations, no such constraints on energetics are available. Assays based on inhibition of growth are indirect and qualitative. Experimental approaches that quantitatively probe interaction free energies are typically based on bulk measurements that average over all faces …