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

TARRAGA, WILSON; FALOMIR LISANDRO; GARDA, H. A.; GONZALEZ MARINA

San Luis

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

Sociedad Argentina de Biofísica SAB

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 arrangement which depend 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, 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 the association constants (Kas) corresponding to the different oligomerization events proposed.The labeled mutants were stable in solution as indicated by 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, indicating the participation of helices 5 and 10 in the contact regions during certain oligomerization. Changes in p-value of monomer emission also reported conformational changes during apo A I oligomerization. In the case of K133C, we predicted two different events of oligomerization in a progressive association model. These results suggest that the proximity in the analyzed positions are necessary to form dHDL in a correct orientation.