Studies with pyrenyl-labeled apolipoprotein A-I. A multiparametric analysis of Pyrenyl-maleimide

TARRAGA WILSON ALBERTO; GONZALEZ MARINA CECILIA; FALOMIR LOCKHART, LISANDRO JORGE; GARDA HORACIO ALBERTO

San Luis

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Sociedad Argentina de Biofísica

Apolipoprotein A‑I (apo A‑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. Apo A‑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, apo A‑I self‑aggregates to form dimers and oligomers of higher orders, with a mechanism unwell characterized. It also interacts with phospholipids and forms discoidal HDL (dHDL) in different anti‑parallel helical arrangements which differ on the proximity of its helices from each other.The aim of the present study is to obtain information on the apo A‑I self‑aggregation in solution, especially helices proximities, which may be important for understanding the mechanisms of dHDL generation.Six cysteine mutants (K107C, K133C F104C, L137C, K226C and F225C) were specifically designed and labeled with pyrenyl‑maleimide in positions corresponding to hydrophilic and hydrophobic faces of helices 4, 5 and 10. The monomer and excimer fluorescence of the labeled proteins were registered as a function of total apo A‑I concentration; and several mathematical models were developed and compared to evaluate the different association types and calculate association constants (Kas) corresponding to the different oligomerization events proposed.The labeled mutants were stable in solution, as indicated by its tryptophan fluorescence. With the exception of F104C, they were biologically active since they can interact with phospholipids to form dHDL. Fluorescence emission spectra of pyrene showed excimer formation only in the case of labeled F225C, K133C and K226C mutants, highlighting the participation of helices 5 and 10 in the contact regions during certain oligomerization steps. Changes in p-value of monomer emission also reported conformational changes during apo A‑I oligomerization. In the case of K133C, we predicted at least two different events of oligomerization, and a model of progressive association seems to be the most suitable to represent this behavior.Altogether, these results suggest that the self-proximities of helixes 5 and 10, which are necessary to form dHDL, are already present in the soluble conformers of apo A-I. AcknowldegmentsThis work was financed by: CONICET, ANPCyT and UNIVERSIDAD NACIONAL DE LA PLATA (UNLP).