Phosphorylation and Ionic Strength Alter the LRAP鈥揌AP Interface in the N-Terminus

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• 作者：Jun-xia Lu ; Yimin Sharon Xu ; Wendy J. Shaw
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：April 2, 2013
• 年：2013
• 卷：52
• 期：13
• 页码：2196-2205
• 全文大小：463K
• 年卷期：v.52,no.13(April 2, 2013)
• ISSN：1520-4995

文摘

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: L¹⁵ to V¹⁹, V¹⁹ to L²³, and K²⁴ to S²⁸. These regions are also near the only phosphorylated residue in the protein pS¹⁶; therefore, changes in the LRAP鈥揌AP interaction as a function of phosphorylation (LRAP(鈭扨) 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 L¹⁵(+P) and K²⁴(鈭扨). The structure and orientation of the LRAP鈥揌AP 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 L¹⁵V¹⁹(+P) did not change significantly as a function of pH or ionic strength. The structure and orientation of the region V¹⁹L²³(+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(鈭扨) and LRAP(+P) as a function of pH or ionic strength, and also reveal that K²⁴ has multiple resolvable resonances, suggestive of two coexisting structures. Phosphorylation also alters the LRAP鈥揌AP interface. All of the three residues investigated (L¹⁵, V¹⁹, and K²⁴) are closer to the surface in LRAP(+P), but only K²⁴S²⁸ changes structure as a result of phosphorylation, from a random coil to a largely helical structure, and V¹⁹L²³ 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.