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首页> 外文期刊>Biochemistry >Ionization Properties of Phosphatidylinositol Polyphosphates in Mixed Model Membranes
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Ionization Properties of Phosphatidylinositol Polyphosphates in Mixed Model Membranes

机译：混合模型膜中磷脂酰肌醇多磷酸盐的电离特性

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ABSTRACT: Phosphatidylinositol polyphosphate lipids (phosphoinositides) form only a minor pool ofmembranenphospholipids but are involved in many intracellular signaling processes, including membrane trafficking,ncytoskeletal remodeling, and receptor signal transduction. Phosphoinositide properties are largely determined bynthe characteristics of their headgroup, which at physiological pH is highly charged but also capable of formingnhydrogen bonds. Many proteins have developed special binding domains that facilitate specific binding tonparticular phosphoinositides, while other proteins interact with phosphoinositides via nonspecific electrostaticninteractions. Despite its importance, only limited information is available about the ionization properties ofnphosphoinositides. We have investigated the pH-dependent ionization behavior of all three naturally occurringnphosphatidylinositol bisphosphates as well as of phosphatidylinositol 3,4,5-trisphosphate in mixed phospho-ninositide/phosphatidylcholine vesicles using magic angle spinning 31nP NMR spectroscopy. For phosphatidyl-ninositol 3,5-bisphosphate, where the two phosphomonoester groups are separated by a hydroxyl group at then4-position, the pH-dependent chemical shiftvariationcanbefittedwithaHenderson-Hasselbalch-typenformalism, yielding pKa2 values of 6.96 ( 0.04 and 6.58 ( 0.04 for the 3- and 5-phosphates, respectively. Inncontrast, phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] as well as phosphatidylinositol 4,5-bisphosphaten[PI(4,5)P2] show a biphasic pH-dependent ionization behavior that cannot be explained by a Hender-nson-Hasselbalch-type formalism. This biphasic behavior can be attributed to the sharing of the last remainingnproton between the vicinal phosphomonoester groups. At pH 7.0, the overall charge (including the phospho-ndiester group charge) is found to be -3.96 ( 0.10 for PI(3,4)P2 and -3.99 ( 0.10 for PI(4,5)P2.WhilefornPI(3,5)P2 and PI(4,5)P2 the charges of the individual phosphate groups in the molecule differ, they are equal fornPI(3,4)P2. Differences in the charges of the phosphomonoester groups can be rationalized on the basis of thenability of the respective phosphomonoester group to form intramolecular hydrogen bonds with adjacentnhydroxyl groups. Phosphatidylinositol 3,4,5-trisphosphate shows an extraordinary complex ionization behavior.nWhile at pH 4 then31nP NMR peak of the 4-phosphate is found downfield from the other two phosphomonoesterngroup peaks, an increase in pH leads to a crossover of the 4-phosphate, which positions this peak eventuallynupfield from the other two peaks. As a result, the 4-phosphate group shows a significantly lower charge at pHnvalues between 7 and 9.5 than the other two phosphomonoester groups. The charge of the respectivenphosphomonoester group in PI(3,4,5)P3 is lower than the corresponding charge of the phosphatidylinositolnbisphosphate phosphomonoester groups, leading to an overall charge of PI(3,4,5)P3 of -5.05 ( 0.15 at pH 7.0.nThe charge of all investigated phosphoinositides at pH 7.0 is equal or higher than the corresponding charge ofnsoluble inositol polyphosphate headgroup analogues, which is the opposite of what is expected on the basis ofnsimple electrostatic considerations. This higher than expected headgroup charge can be rationalized with mutualnintermolecular hydrogen bond formation.Measurements using different concentrations of PI(4,5)P2 in the lipidnvesicles (1, 5, and 20 mol %) did not reveal any significant concentration-dependent shift of the twonphosphomonoester peaks, suggesting that PI(4,5)P2 isclusteredevenat1mol%.
机译：摘要：磷脂酰肌醇多磷酸脂（磷酸肌醇）仅形成少量的膜磷脂，但参与许多细胞内信号传导过程，包括膜运输，细胞骨架重塑和受体信号转导。磷脂酰肌醇的性质主要取决于其首基的特征，该首基在生理pH下是高电荷的，但也能够形成氢键。许多蛋白质已开发出特殊的结合结构域，可促进特异性结合特定的磷酸肌醇，而其他蛋白质则通过非特异性静电相互作用与磷酸肌醇相互作用。尽管它很重要，但是关于正磷酸肌醇离子化性质的信息很少。我们已经使用魔角旋转31nP NMR光谱技术研究了所有三种天然存在的磷脂酰肌醇双磷酸酯以及磷脂酰肌醇3,4,5-三磷酸酯的pH依赖性电离行为。对于磷脂酰肌醇3,5-二磷酸酯，其中两个磷酸单酯基团在然后的4位被羟基隔开，pH依赖的化学位移变化可以用亨德森-哈塞尔巴尔奇式的形式主义进行拟合，得出pKa2值为6.96（0.04和6.58（0.04为分别为3-和5-磷酸酯，Inntrast，磷脂酰肌醇3,4-双磷酸酯[PI（3,4）P2]以及磷脂酰肌醇4,5-双磷酸酯[PI（4,5）P2]显示出两相pH依赖性Hender-nson-Hasselbalch型形式主义无法解释的电离行为。这种两相行为可归因于相邻的磷酸单酯基团之间共享最后一个剩余的质子。在pH 7.0时，总电荷（包括磷酸二酯基团）电荷）为-3.96（PI（3,4）P2为0.10和-3.99（PI（4,5）P2为0.10.WhilefornPI（3,5）P2和PI（4,5）P2分子中的各个磷酸基团不同，它们对于nPI（3,4）P2是相同的。可以基于相应的磷酸单酯基团随后与相邻的羟基形成分子内氢键的能力来合理化磷酸单酯基团的电荷。磷脂酰肌醇3,4,5-三磷酸酯显示出非凡的复杂电离行为.n虽然在pH 4时从其他两个磷酸单酯基团峰的低场发现了4-磷酸盐的NMR峰，但pH的升高导致4-磷酸盐的交换，该峰最终位于其他两个峰的正上方。结果，与其他两个磷酸单酯基相比，4-磷酸基在pHn值介于7和9.5之间时显示出明显更低的电荷。 PI（3,4,5）P3中相应的正磷酸单酯基团的电荷低于磷脂酰肌醇双磷酸磷酸单酯基团的相应电荷，导致PI（3,4,5）P3的总电荷为-5.05（pH为0.15 7.0.n在pH 7.0时所有研究的磷酸肌醇的电荷等于或高于相应的不溶性肌醇多磷酸头基类似物的电荷，这与基于简单的静电考虑而预期的相反，该高于预期的头基电荷可通过以下方法合理化分子间氢键的形成。使用脂质囊泡中浓度不同的PI（4,5）P2（1、5和20 mol％）进行的测量未发现二磷酸单酯峰的任何明显的浓度依赖性变化，表明PI（4， 5）P 2聚集为1mol％。

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