Department of Chemistry and Biochemistry, 1250 Bellflower Boulevard, California State University Long Beach, Long Beach, California 90840, USA.
Biochemistry. 2010 Mar 2;49(8):1766-75. doi: 10.1021/bi901902e.
The C-terminal domain (CT) of apolipoprotein E (apoE), a critical protein involved in cholesterol transport in the plasma and brain, plays an important role in high-affinity lipoprotein binding. Although high-resolution structural information is available for the N-terminal domain of apoE, the structural organization of the CT (residues 201-299) is largely unknown. In this study, we employ site-specific fluorescence labeling with pyrene maleimide to gain insight into the structure and conformation of apoE CT in its naturally self-associated state in buffer at physiologically relevant concentrations (5-50 microg/mL). Pyrene is a highly sensitive fluorophore that reports on spatial proximity between desired sites by displaying unique spectral features. Pyrene was covalently attached to single cysteine-containing recombinant human apoE CT at position 223 or 255 to probe the first predicted helical segment and at position 277 to monitor the terminal predicted helical segment. Regardless of the location of the probe, all three pyrene-labeled apoE CT variants display an intense and dramatic fluorescence excimer band at 460 nm, a signature feature of pyrene, which indicates that two pyrene moieties are within 10 A of each other. In addition, an intense peak at 387 nm (indicative of a highly hydrophobic environment) was noted in all cases. Fluorescence emission quenching by potassium iodide indicates that the accessibility to the probes was restricted at these locations. The possibility that the hydrophobicity of the pyrene moiety was the driving force for helix-helix interaction was excluded because pyrene located at position 209, which is predicted to be located in a nonhelical segment, did not display the above intense unique features. Lastly, denaturation studies suggest that the terminal helix unfolds prior to the first predicted helix in apoE CT. Our studies indicate that there are extensive intermolecular helix-helix contacts throughout the entire CT in the lipid-free state with two apoE CT molecules oriented parallel to each other to form a dimer, which dimerizes further to yield a tetramer. Such an organization allows helix-helix interactions to be replaced by helix-lipid interactions upon encountering a lipoprotein surface, with the terminal helix likely initiating the binding interaction. This study presents the possibility of employing pyrene fluorophores as powerful new alternatives to obtain conformational information of proteins at physiologically relevant concentrations.
载脂蛋白 E (apoE) 的 C 端结构域 (CT) 是一种参与血浆和大脑中胆固醇转运的关键蛋白，在高亲和力脂蛋白结合中发挥重要作用。尽管 apoE 的 N 端结构域具有高分辨率的结构信息，但 CT（残基 201-299）的结构组织在很大程度上是未知的。在这项研究中，我们采用特异性荧光标记法，使用芘马来酰亚胺，深入了解 apoE CT 在生理相关浓度（5-50μg/ml）下自然自缔合状态下的结构和构象。芘是一种高灵敏度的荧光团，通过显示独特的光谱特征来报告所需位点之间的空间接近度。芘通过共价连接到重组人 apoE CT 的位置 223 或 255 处的单个半胱氨酸来探测第一个预测的螺旋片段，并连接到位置 277 处以监测末端预测的螺旋片段。无论探针的位置如何，所有三种芘标记的 apoE CT 变体在 460nm 处都显示出强烈而显著的荧光激基缔合物带，这是芘的特征特征，表明两个芘部分彼此之间的距离在 10Å 以内。此外，在所有情况下都注意到 387nm 处的强峰（指示高度疏水性环境）。碘化钾的荧光发射猝灭表明在这些位置探针的可及性受到限制。芘部分的疏水性是螺旋-螺旋相互作用驱动力的可能性被排除，因为位于预测位于非螺旋片段的位置 209 的芘部分没有显示出上述强烈的独特特征。最后，变性研究表明，apoE CT 中的末端螺旋在第一个预测的螺旋之前展开。我们的研究表明，在无脂状态下，整个 CT 中存在广泛的分子间螺旋-螺旋接触，两个 apoE CT 分子彼此平行排列形成二聚体，进一步二聚化形成四聚体。这种组织允许在遇到脂蛋白表面时，将螺旋-螺旋相互作用替换为螺旋-脂质相互作用，末端螺旋可能启动结合相互作用。这项研究提出了一种可能性，即可以使用芘荧光团作为获得在生理相关浓度下蛋白质构象信息的有力新替代方法。
