Pacific Northwest National Laboratory, Richland, WA 99354, USA.
Biochemistry. 2013 Apr 2;52(13):2196-205. doi: 10.1021/bi400071a. Epub 2013 Mar 22.
The conditions present during enamel crystallite development change dramatically as a function of time, including the pH, protein concentration, surface type, and ionic strength. In this work, we investigate the role that two of these changing conditions, pH and ionic strength, have in modulating the interaction of the amelogenin, LRAP, with hydroxyapatite (HAP). Using solid-state NMR dipolar recoupling and chemical shift data, we investigate the structure, orientation, and dynamics of three regions in the N-terminus of the protein: L(15) to V(19), V(19) to L(23), and K(24) to S(28). These regions are also near the only phosphorylated residue in the protein pS(16); therefore, changes in the LRAP-HAP interaction as a function of phosphorylation (LRAP(-P) vs LRAP(+P)) were also investigated. All of the regions and conditions studied for the surface immobilized proteins showed restricted motion, with indications of slightly more mobility under all conditions for L(15)(+P) and K(24)(-P). The structure and orientation of the LRAP-HAP interaction in the N-terminus of the phosphorylated protein is very stable to changing solution conditions. From REDOR dipolar recoupling data, the structure and orientation in the region L(15)V(19)(+P) did not change significantly as a function of pH or ionic strength. The structure and orientation of the region V(19)L(23)(+P) were also stable to changes in pH, with the only significant change observed at high ionic strength, where the region becomes extended, suggesting this may be an important region in regulating mineral development. Chemical shift studies also suggest minimal changes in all three regions studied for both LRAP(-P) and LRAP(+P) as a function of pH or ionic strength, and also reveal that K(24) has multiple resolvable resonances, suggestive of two coexisting structures. Phosphorylation also alters the LRAP-HAP interface. All of the three residues investigated (L(15), V(19), and K(24)) are closer to the surface in LRAP(+P), but only K(24)S(28) changes structure as a result of phosphorylation, from a random coil to a largely helical structure, and V(19)L(23) becomes more extended at high ionic strength when phosphorylated. These observations suggest that ionic strength and dephosphorylation may provide switching mechanisms to trigger a change in the function of the N-terminus during enamel development.
在釉质晶体生长过程中，随着时间的推移，其所处环境的条件会发生显著变化，包括 pH 值、蛋白质浓度、表面类型和离子强度。在这项工作中，我们研究了其中两个变化条件（pH 值和离子强度）对釉原蛋白（LRAP）与羟基磷灰石（HAP）相互作用的调节作用。我们使用固态 NMR 偶极重聚和化学位移数据，研究了蛋白质 N 端三个区域的结构、取向和动力学：L(15)到 V(19)、V(19)到 L(23)和 K(24)到 S(28)。这些区域也靠近蛋白质中唯一的磷酸化残基 pS(16)；因此，我们还研究了磷酸化（LRAP(-P) 与 LRAP(+P)）对 LRAP-HAP 相互作用的影响。所有研究的表面固定化蛋白的区域和条件都表现出受限的运动，在所有条件下，L(15)(+P)和 K(24)(-P)的运动性略高。磷酸化蛋白 N 端 LRAP-HAP 相互作用的结构和取向对溶液条件的变化非常稳定。从 REDOR 偶极重聚数据可以看出，在 pH 值或离子强度变化时，L(15)V(19)(+P) 区域的结构和取向没有明显变化。在 pH 值变化时，V(19)L(23)(+P) 区域的结构和取向也很稳定，只有在高离子强度下才观察到明显变化，此时该区域变得伸展，这表明这可能是调节矿物质发育的重要区域。化学位移研究还表明，对于 LRAP(-P)和 LRAP(+P)，在 pH 值或离子强度变化时，所有三个研究区域的结构都没有明显变化，并且还表明 K(24)具有多个可分辨的共振峰，提示存在两种共存结构。磷酸化还改变了 LRAP-HAP 界面。在 LRAP(+P)中，所有三个研究的残基（L(15)、V(19)和 K(24)）都更接近表面，但只有 K(24)S(28)的结构因磷酸化而发生变化，从无规卷曲转变为大部分螺旋结构，而 V(19)L(23)在高离子强度下变得更加伸展。这些观察结果表明，离子强度和去磷酸化可能提供了一种开关机制，以在釉质发育过程中触发 N 端功能的变化。
