Lee Jung Ho, Ying Jinfa, Bax Ad
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , Bethesda, Maryland 20892, United States.
Biochemistry. 2016 Sep 6;55(35):4949-59. doi: 10.1021/acs.biochem.6b00637. Epub 2016 Aug 23.
The interaction between α-synuclein (αS) protein and lipid membranes is key to its role in synaptic vesicle homeostasis and plays a role in initiating fibril formation, which is implicated in Parkinson's disease. The natural state of αS inside the cell is generally believed to be intrinsically disordered, but chemical cross-linking experiments provided evidence of a tetrameric arrangement, which was reported to be rich in α-helical secondary structure based on circular dichroism (CD). Cross-linking relies on chemical modification of the protein's Lys C(ε) amino groups, commonly by glutaraldehyde, or by disuccinimidyl glutarate (DSG), with the latter agent preferred for cellular assays. We used ultra-high-resolution homonuclear decoupled nuclear magnetic resonance experiments to probe the reactivity of the 15 αS Lys residues toward N-succinimidyl acetate, effectively half the DSG cross-linker, which results in acetylation of Lys. The intensities of both side chain and backbone amide signals of acetylated Lys residues provide direct information about the reactivity, showing a difference of a factor of 2.5 between the most reactive (K6) and the least reactive (K102) residue. The presence of phospholipid vesicles decreases reactivity of most Lys residues by up to an order of magnitude at high lipid:protein stoichiometries (500:1), but only weakly at low ratios. The decrease in Lys reactivity is found to be impacted by lipid composition, even for vesicles that yield similar αS CD signatures. Our data provide new insight into the αS-bilayer interaction, including the pivotal state in which the available lipid surface is limited. Protection of Lys C(ε) amino groups by αS-bilayer interaction will strongly impact quantitative interpretation of DSG cross-linking experiments.
α-突触核蛋白（αS）与脂质膜之间的相互作用是其在突触小泡稳态中发挥作用的关键，并且在引发原纤维形成过程中起作用，而原纤维形成与帕金森病有关。一般认为细胞内αS的天然状态是内在无序的，但化学交联实验提供了四聚体排列的证据，基于圆二色性（CD），据报道这种排列富含α-螺旋二级结构。交联依赖于蛋白质赖氨酸C（ε）氨基的化学修饰，通常使用戊二醛，或使用戊二酸二琥珀酰亚胺酯（DSG），后者更适合用于细胞分析。我们使用超高分辨率同核去耦核磁共振实验来探测15个αS赖氨酸残基对乙酸N-琥珀酰亚胺酯（实际上是DSG交联剂的一半）的反应性，这会导致赖氨酸乙酰化。乙酰化赖氨酸残基的侧链和主链酰胺信号强度提供了关于反应性的直接信息，显示出最具反应性的残基（K6）和最不具反应性的残基（K102）之间相差2.5倍。在高脂质：蛋白质化学计量比（500:1）下，磷脂囊泡的存在使大多数赖氨酸残基的反应性降低多达一个数量级，但在低比例时影响较弱。发现赖氨酸反应性的降低受脂质组成的影响，即使对于产生相似αS CD特征的囊泡也是如此。我们的数据为αS-双层相互作用提供了新的见解，包括可用脂质表面受限的关键状态。αS-双层相互作用对赖氨酸C（ε）氨基的保护将强烈影响DSG交联实验的定量解释。
