赖氨酸残基控制β 2-糖蛋白I的构象动力学
Lysine residues control the conformational dynamics of beta 2-glycoprotein I
摘要：
蛋白质动力学研究中的一个主要问题是改变构象的可视化，这对从酶催化到信号传导的过程都很重要。表现出构象动力学的蛋白质是可溶性血液蛋白β 2-糖蛋白I（β 2GPI），其以两种构象存在：闭合（环状）形式和开放（线性）形式。据推测，开放构象的比例增加导致自身免疫性疾病抗磷脂综合征（APS）。beta2 GPI的一个特征是赖氨酸残基含量高。然而，赖氨酸在β 2GPI构象动力学中的潜在作用研究得很少。在这里，我们报告的战略，永久打开了封闭的蛋白质构象的化学乙酰化赖氨酸残基使用乙酸N-羟基琥珀酰亚胺酯（NHS-Ac）。通过用氟醛邻苯二甲醛（OPA）试剂定量伯氨基以及用抗乙酰化赖氨酸抗体进行蛋白质印迹分析来证明特异性和完全乙酰化。我们的研究结果表明，乙酰化beta2 GPI保留其二级和三级结构，如圆二色光谱所示。我们发现，赖氨酸乙酰化后，大多数蛋白质是在开放的构象所揭示的原子力显微镜高分辨率图像。使用这种策略，我们证明了赖氨酸残基的静电相互作用在稳定beta2 GPI闭合构象中起着重要作用，正如赖氨酸电荷分布计算所证实的那样。我们预见，我们的方法将适用于其他富含赖氨酸的蛋白质（如组蛋白）经历构象转变。
英文摘要：
One of the major problems in the study of the dynamics of proteins is the visualization of changing conformations that are important for processes ranging from enzyme catalysis to signaling. A protein exhibiting conformational dynamics is the soluble blood protein beta 2-glycoprotein I (beta2GPI), which exists in two conformations: the closed (circular) form and the open (linear) form. It is hypothesized that an increased proportion of the open conformation leads to the autoimmune disease antiphospholipid syndrome (APS). A characteristic feature of beta2GPI is the high content of lysine residues. However, the potential role of lysine in the conformational dynamics of beta2GPI has been poorly investigated. Here, we report on a strategy to permanently open up the closed protein conformation by chemical acetylation of lysine residues using acetic acid N-hydroxysuccinimide ester (NHS-Ac). Specific and complete acetylation was demonstrated by the quantification of primary amino groups with fluoraldehyde o-phthalaldehyde (OPA) reagent, as well as western blot analysis with an anti-acetylated lysine antibody. Our results demonstrate that acetylated beta2GPI preserves its secondary and tertiary structures, as shown by circular dichroism spectroscopy. We found that after lysine acetylation, the majority of proteins are in the open conformation as revealed by atomic force microscopy high-resolution images. Using this strategy, we proved that the electrostatic interaction of lysine residues plays a major role in stabilizing the beta2GPI closed conformation, as confirmed by lysine charge distribution calculations. We foresee that our approach will be applied to other lysine-rich proteins (e.g. histones) undergoing conformational transitions.
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