Naganagowda G A, Gururaja T L, Levine M J
Department of Oral Biology and Dental Research Institute, State University of New York at Buffalo, 14214-3092, USA.
J Biomol Struct Dyn. 1998 Aug;16(1):91-107. doi: 10.1080/07391102.1998.10508230.
Membrane-induced solution structure of human salivary statherin, a 43 amino acid residue acidic phosphoprotein, has been investigated by two-dimensional proton nuclear magnetic resonance (2D 1H NMR) spectroscopy. NMR assignments and structural analysis of this phosphoprotein was accomplished by analyzing the pattern of sequential and medium range NOEs, alphaCH chemical shift perturbations and deuterium exchange measurements of the amide proton resonances. The NMR data revealed three distinct structural motifs in the molecule: (1) an alpha-helical structure at the N-terminal domain comprising Asp1-Tyr16, (2) a polyproline type II (PPII) conformation predominantly occurring at the middle proline-rich domain spanning Gly19-Gln35, and (3) a 3(10)-helical structure at the C-terminal Pro36-Phe43 sequence. Presence of a few weak dalphaN(i,i+2) NOEs suggests that N-terminus also possesses minor population of 3(10)-helical conformation. Of the three secondary structural elements, helical structure formed by the N-terminal residues, Asp1-Ile11 appears to be more rigid as observed by the relatively very slow exchange of amide hydrogens of Glu5-Ile11. 31P NMR experiments clearly indicated that N-terminal domain of statherin exists mainly in disordered state in water whereas, upon addition of structure stabilizing co-solvent, 2,2,2-trifluorethanol (TFE), it showed a strong propensity for helical conformation. Calcium ion interaction studies suggested that the disordered N-terminal region encompassing the two vicinal phosphoserines is essential for the binding of calcium ions in vivo. Results from the circular dichroism (CD) experiments were found to be consistent with and complimentary to the NMR data and provided an evidence that non-aqueous environment such as TFE, could induce the protein to fold into helical conformation. The findings that the statherin possesses blended solvent sensitive secondary structural elements and the requirement of non-structured N-terminal region under aqueous environment in calcium ion interaction may be invaluable to understand various physiological functions of statherin in the oral fluid.
通过二维质子核磁共振（2D 1H NMR）光谱研究了人唾液磷蛋白（一种由43个氨基酸残基组成的酸性磷蛋白）的膜诱导溶液结构。通过分析连续和中程核Overhauser效应（NOE）模式、αCH化学位移扰动以及酰胺质子共振的氘交换测量，完成了该磷蛋白的核磁共振归属和结构分析。核磁共振数据揭示了该分子中的三个不同结构基序：（1）N端结构域（包含Asp1-Tyr16）的α螺旋结构；（2）主要出现在富含脯氨酸的中间结构域（跨越Gly19-Gln35）的多聚脯氨酸II型（PPII）构象；（3）C端Pro36-Phe43序列的3(10)螺旋结构。存在一些较弱的dαN(i,i+2) NOE表明N端也存在少量的3(10)螺旋构象。在这三个二级结构元件中，由N端残基Asp1-Ile11形成的螺旋结构似乎更刚性，这可通过Glu5-Ile11酰胺氢相对非常缓慢的交换观察到。31P NMR实验清楚地表明，在水中，磷蛋白的N端结构域主要以无序状态存在，而加入结构稳定共溶剂2,2,2-三氟乙醇（TFE）后，它显示出强烈的螺旋构象倾向。钙离子相互作用研究表明，包含两个相邻磷酸丝氨酸的无序N端区域对于体内钙离子的结合至关重要。圆二色性（CD）实验结果与核磁共振数据一致且相互补充，证明了非水环境（如TFE）可诱导蛋白质折叠成螺旋构象。磷蛋白具有混合溶剂敏感的二级结构元件以及在水相环境中钙离子相互作用时对非结构化N端区域的需求，这一发现对于理解磷蛋白在口腔液体中的各种生理功能可能具有重要价值。
