Selfassociation of apo AI study with multipleassociation models: A multiparametric analysis of Pyrenylmaleimide

FALOMIR LOCKHART LJ; GONZALEZ MC; TARRAGA W; GARDA HA

Reunión virtual

Jornada; Primeras Jornadas Virtuales de la Sociedad Argentina de Biofísica; 2020

Sociedad Argentina de Biofísica (SAB)

Apolipoprotein AI (apo AI) is the main protein of high-density lipoproteins (HDL), to whichantiatherogenic properties are attributed to its role in the reverse transport of cholesterol excessfrom peripheral tissues to the liver for catabolism and disposal.Apo AI is composed of several amphipathic alphahelices. In water solution, they form a bundle withpoorly characterized tertiary and quaternary structures. Depending on the concentration, apo AIselfaggregates to form dimers and oligomers of higher orders, with a mechanism unwellcharacterized. It also interacts with phospholipids and forms discoidal HDL (dHDL) in differentantiparallel helical arrangement which differ on the proximity of its helices from each other.The aim of the present study is to obtain information on the apo AI selfaggregation in solution,especially helices proximities, which may be important for understanding the mechanisms of dHDLgeneration.Cysteine mutants of apo AI (K107C, K133C and K226C), which reported spectral pyrene´s changesby self-proximity and environmental changes around the probe, were purified and labelled withpyrenylmaleimide in positions corresponding to helices 4 (K107C), 5 (K133C) and 10 (F225C). Themonomer and excimer fluorescence of the labelled proteins were registered as a function of totalapo AI concentration and different multiple-association models were developed to compared andevaluate the different association types, and calculate association constants (K A s) corresponding tothe different oligomerization events proposed.The labelled mutants were stable in solution, as indicated by its tryptophan fluorescence. Theywere biologically active since they can interact with phospholipids to form dHDL. Fluorescenceemission spectra of pyrene showed excimer formation only in the case of labelled K133C and F225Cmutants and P-value environmental changes with all of them, highlighting the participation ofhelices 5 and 10 in the contact regions during certain oligomerization steps. We predicted at leasttwo different events of oligomerization, with the exception of K107C, and a model of progressiveassociation seems to be the most suitable to represent these behaviors. The result reported heresuggest that the selfassociation of apo AI could be initiated by H5 selfinteractions and followed byH10 selfinteractions. Altogether, these results suggest that these selfproximities, which arenecessary to form dHDL, are already present in the soluble conformers of apo AI.AcknowldegmentsThis work was financed by: CONICET, ANPCyT and UNIVERSIDAD NACIONAL DE LA PLATA (UNLP).