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_A) 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.

ApoA-I是HDL的主要蛋白，具有抗动脉粥样硬化特性，可归因于胆固醇逆向转运。它与其他可交换载脂蛋白共享高水平的结构可塑性。在无脂状态下，载脂蛋白两亲性α-螺旋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作用。在这项研究中，我们采用了多参数荧光探针来研究apoA-I的自缔合。我们构建了六个单半胱氨酸突变体，它们沿三个螺旋分布：F104C，K107C（H4），K133C，L137C（H5），F225C和K226C（H10）；并用N-马来酰亚胺P标记。利用其光谱特性，即形成激发的二聚体（准分子）和其荧光精细结构的极性相关变化（P值），我们以无脂形式监测apoA-I自缔合与其浓度的关系。准分子发射突出了H5（K133C）和H10（F225C和K226C）螺旋中的相互作用。而在螺旋H4（K107C）以及螺旋H5和H10中报告了极性变化。开发了数学模型以丰富数据分析并估计关联常数（K _A）和低聚物种分布。此外，我们简要讨论了即使在单个标记位置，多参数荧光探针也可用于监视不同的平衡。结果表明，必须考虑apoA-I自缔合才能充分了解其生理作用。特别地，稳定盘状HDL颗粒的一些接触似乎已经存在于无脂质的apoA-I低聚物中。