Self-association of Apolipoprotein A-I study with multiple-association models: A Pyrene Multiparametric analysis.

GONZALEZ MARINA CECILIA; FALOMIR LOCKHART, LISANDRO J.; TÁRRAGA, WILSON A.; GARDA, HORACIO A.

Jornada; Primeras jornadas virtuales de la Sociedad Argentina de Biofísica; 2020

Sociedad Argentina de Biofísica SAB

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoproteins (HDL), to which antiatherogenic properties are attributed to its role in the reverse transport of cholesterol excess from peripheral tissues to the liver for catabolism and disposal.ApoA-I is composed of several amphipathic alpha-helices. In water solution, they form a bundle with poorly characterized tertiary and quaternary structures. Depending on the concentration, apoA-I self-aggregates form dimers and oligomers of higher orders, with a mechanism unwell characterized. It also interacts with phospholipids and forms discoidal HDL (dHDL) in alternative anti-parallel helical arrangements which differ on the proximity of its helices from each other.The aim of the present study is to characterize apoA I oligomerization in solution, focusing on the proximity of helices known to be important for dHDL formation and stabilization.Fluorescently labelled variants of apoA-I have previously been reported to be sensitive to its self association. Selected cysteine mutants were purified and labelled with pyrenyl-maleimide in positions corresponding to helices 4 (K107C), 5 (K133C) and 10 (F225C). The monomer and excimer fluorescence of the labelled proteins were registered as a function of total apoA I concentration. Different multiple association models were developed to compare and evaluate alternative oligomerization schemes, and to estimate the association constants (KA) corresponding to individual association events.The labelled mutants were stable in solution, as indicated by its tryptophan fluorescence and denaturation curves. They were biologically active since they can form dHDL. Fluorescence emission spectra of pyrene showed excimer formation only in the case of labelled K133C and F225C variants; and P-value environmental changes was also observed in all mutants, highlighting the participation of helices 5 and 10 in the contact regions during certain oligomerization steps. We predicted at least two different events of oligomerization, and a model of progressive association seems to be the most suitable to represent these behaviors. The results reported here suggest that the self-association of apoA I could be initiated by H5 self-interactions and followed by H10 self interactions. Altogether, these results suggest that these self-proximities, which are may also be critical to form dHDL, are already present in the soluble conformers of apoA I.