Multiparametric analysis of Pyrenyl-maleimide labeled human apolipoprotein A‑I cystein mutants

TÁRRAGA WA; GONZALEZ MC; FALOMIR LOCKHART LJ; GARDA HA

La Plata

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Argentina de Biofísica

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 reversetransport of cholesterol excess from peripheral tissues to the liver for catabolismand disposal.ApoA-I is composed of several amphipathic alpha-helices. In water solution, theyform a bundle with poorly characterized tertiary and quaternary structures.Depending on the concentration, apo A-I self-aggregates to form dimers andoligomers of higher orders. It also interacts with phospholipids and forms discoidalHDL (dHDL).The aim of the present study is to obtain information on the apoA-I selfaggregationin solution important for understanding the mechanisms of HDLgeneration. Six cysteine mutants (K107C, K133C F104C, L137C, K226C and F225C)were specifically designed and labeled with pyrenyl‑maleimide in positionscorresponding to hydrophilic and hydrophobic faces of helices 4, 5 and 10. Themonomer and excimer fluorescence of the labeled proteins were registered as afunction of total apoA‑I concentration; and several mathematical models weredeveloped and compared to evaluate dissociation constants (Kds) correspondingto the different oligomerization events.The labeled mutants were stable in solution as indicated by tryptophanfluorescence. With the exception of F104C, they were biologically active since theycan interact with phospholipids to form dHDL. Fluorescence emission spectra ofpyrene showed excimer formation only in the case of labeled F225C, K133C andK226C mutants, indicating the participation of helices 5 and 10 in the contactregions during certain oligomerization. Changes in p-value of monomer emissionalso reported conformational changes during apoA‑I oligomerization.