Instituto de Investigaciones Bioquímica de La Plata (INIBIOLP), Centro Científico Tecnológico-La Plata, Calle 60 y 120 s/n, 1900, La Plata, Argentina.
Instituto de Investigaciones Bioquímica de La Plata (INIBIOLP), Centro Científico Tecnológico-La Plata, Calle 60 y 120 s/n, 1900, La Plata, Argentina; Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Calle 47 y 115 s/n, 1900, La Plata, Argentina.
Arch Biochem Biophys. 2021 Mar 15;699:108748. doi: 10.1016/j.abb.2020.108748. Epub 2021 Jan 12.
ApoA-I is the main protein of HDL which has anti-atherogenic properties attributed to reverse cholesterol transport. It shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and inter-molecularly, providing structural stabilization by a complex self-association mechanism. In this study, we employed a multi-parametric fluorescent probe to study the self-association of apoA-I. We constructed six single cysteine mutants spanning positions along three helices: F104C, K107C (H4), K133C, L137C (H5), F225C and K226C (H10); and labelled them with N-Maleimide Pyrene. Taking advantage of its spectral properties, namely formation of an excited dimer (excimer) and polarity-dependent changes in its fluorescence fine structure (P-value), we monitored the apoA-I self-association in its lipid-free form as a function of its concentration. Interactions in helices H5 (K133C) and H10 (F225C and K226C) were highlighted by excimer emission; while polarity changes were reported in helix H4 (K107C), as well as in helices H5 and H10. Mathematical models were developed to enrich data analysis and estimate association constants (K) and oligomeric species distribution. Furthermore, we briefly discuss the usefulness of the multi-parametric fluorescent probe to monitor different equilibria, even at a single labelling position. Results suggest that apoA-I self-association must be considered to fully understand its physiological roles. Particularly, some contacts that stabilize discoidal HDL particles seem to be already present in the lipid-free apoA-I oligomers.
载脂蛋白 A-I 是 HDL 的主要蛋白质，具有抗动脉粥样硬化特性，归因于胆固醇逆转运。它与其他可交换载脂蛋白一样，具有高水平的结构可塑性。在无脂状态下，载脂蛋白的两亲性α-螺旋在分子内和分子间相互作用，通过复杂的自缔合机制提供结构稳定性。在这项研究中，我们使用多参数荧光探针研究了载脂蛋白 A-I 的自缔合。我们构建了六个单半胱氨酸突变体，跨越三个螺旋的位置：F104C、K107C（H4）、K133C、L137C（H5）、F225C 和 K226C（H10）；并用 N-马来酰亚胺芘对它们进行标记。利用其光谱特性，即形成激发二聚体（激态二聚体）和荧光精细结构的极性依赖性变化（P 值），我们监测了无脂形式的载脂蛋白 A-I 自缔合作为其浓度的函数。在 H5 螺旋（K133C）和 H10 螺旋（F225C 和 K226C）中，激态二聚体发射突出了相互作用；而在 H4 螺旋（K107C）以及 H5 和 H10 螺旋中，报道了极性变化。开发了数学模型来丰富数据分析并估计缔合常数（K）和寡聚体分布。此外，我们简要讨论了多参数荧光探针在监测不同平衡，甚至在单个标记位置的有用性。结果表明，必须考虑载脂蛋白 A-I 的自缔合，以充分理解其生理作用。特别是，一些稳定盘状 HDL 颗粒的接触似乎已经存在于无脂载脂蛋白 A-I 寡聚体中。
