Self-association of Apolipoprotein A-I studied with multiple-association models: A pyrene multiparametric analysis.

Abstract

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein particles, conferring anti-atherogenic properties through reverse cholesterol transport. In its lipid-free state, apoA-I self-associates, a process implicated in normal physiology as well as in pathological conditions, such as amyloidosis. Although various mechanisms have been proposed to explain its self-association, there is still no consensus, and its functional implications remain unclear. Here, we employed a multi-parametric fluorescent probe to investigate apoA-I self-association. We used three single cysteine mutants located in different helixes: K107C (H4), K133C (H5), and F225C (H10); and labelled them with pyrene as detailed previously (Tárraga et al. Arch Biochem Biophys 699 (2021) 108748). Our original experiments revealed excimer emission between helixes H5 and H10 and polarity changes also in H4. By exploiting specific pyrene band emissions to monitor dimer association (via excimer formation) and microenvironment polarity (P-value), we tracked apoA-I oligomerization as a function of protein concentration. Several mathematical models of self-association were developed and compared using selection criteria to identify the simplest model reproducing apoA-I's complex behaviour in aqueous media: a Sequential Association submodel limited to a tetramer as the highest order oligomeric species and considering both dimers and tetramers as responsible for the excimer's emission. Multi-equilibria models enhanced the titration analysis, allowing estimation of association constants (Ka) and oligomeric species distribution. Our results support previous evidence that contacts among helixes, which stabilize discoidal HDL particles, are already present in lipid-free apoA-I, with dimeric species predominating, and possible tetrameric too. Further investigation of these species is essential to elucidate their physiological and pathological roles, such as in atherosclerosis.