A solution NMR investigation into the murine amelogenin splice-variant LRAP (Leucine-Rich Amelogenin Protein)
A solution NMR investigation into the murine amelogenin splice-variant LRAP (Leucine-Rich Amelogenin Protein)
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DOI：
10.1016/j.bbapap.2010.03.006
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发表时间：
2010-09-01
影响因子：
3.2
通讯作者：
Shaw, Wendy J.
中科院分区：
文献类型：
--
作者：
Buchko, Garry W.;Tarasevich, Barbara J.;Shaw, Wendy J.
釉原蛋白是牙釉质生物矿化早期阶段存在于成釉细胞中的主要蛋白质，占基质蛋白的 90% 以上。除了全长蛋白外，还存在几种牙釉蛋白剪接变体亚型，包括 LRAP（富含亮氨酸的牙釉蛋白），该蛋白由全长牙釉蛋白的前 33 个和最后 26 个残基组成。使用溶液态核磁共振波谱，我们通过广泛使用先前对全长鼠牙釉蛋白 (rp(H) M180) 的化学位移分配，在 pH 3.0 的 2% 乙酸中分配了鼠 LRAP (rp(H)LRAP) 的 H-1-N-15 HSQC 谱。这种相关性是可能的，因为 LRAP 与全长蛋白质一样，在这些溶液条件下本质上是无序的。 rp(H)M180 和 rp(H)LRAP 的 H-1-N-15 HSQC 谱之间的主要区别是 rp(H)LRAP N 末端附近的 S12* 和 Y12 之间的七个非脯氨酸残基中的每一个都有一组附加的酰胺共振，表明 LRAP 的 N 末端区域存在两种不同的构象。对脯氨酸碳化学位移的分析表明，这两种状态的分子基础不是 N 末端区域中一个或多个脯氨酸残基的顺反异构化。从 2% 乙酸开始，其中 rp(H)LRAP 在溶液中为单体，添加 NaCl 会影响分子动力学中的残留物特异性变化，表现为 H-1-N-15 HSQC 交叉峰强度的降低和消失。正如对全长蛋白质所观察到的，这些扰动可能发出控制 rp(H)LRAP 超分子自组装成纳米球的早期事件的信号。然而，高盐中 rp(H)LRAP 和 rp(H)M180 之间 H-1-N-15 HSQC 交叉峰扰动的不同模式表明，末端在各自的纳米球中可能表现不同，并且也许这些差异有助于归因于 LRAP 而不是全长蛋白的细胞信号传导特性。 (C) 2010 年爱思唯尔作者。版权所有。
Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up >90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the H-1-N-15 HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H) M180). This correlation was possible because LRAP, like the full-length protein, is intrinsically disordered under these solution conditions. The major difference between the H-1-N-15 HSQC spectra of rp(H)M180 and rp (H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12* and Y12 near the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggests that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region. Starting from 2% acetic acid, where rp(H)LRAP was monomeric in solution, NaCl addition effected residue specific changes in molecular dynamics manifested by the reduction in intensity and disappearance of H-1-N-15 HSQC cross peaks. As observed for the full-length protein, these perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different patterns of H-1-N-15 HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences contribute to the cell signaling properties attributable to LRAP but not to the full-length protein. (C) 2010 Elsevier By. All rights reserved.