Autoagregación de Apolipoproteína A-I Humana. Estudios con mutantes de cisteína marcadas con pirenil maleimida

TARRAGA WILSON ALBERTO

La Plata

Seminario; Seminario-INIBIOLP; 2016

Instituto de Investigaciones Bioquímicas de La Plata "Profesor Doctor Rodolfo R. Brenner"

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, apo A-I self-aggregates to form dimers and oligomers of higher orders. It also interacts with phospholipids and forms discoidal HDL (dHDL).The aim of the present study is to obtain information on the apoA-I self-aggregation in solution important for understanding the mechanisms of HDL 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 apoA I concentration; and several mathematical models were developed and compared to evaluate dissociation constants (Kds) corresponding to the different oligomerization events.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 apoA I oligomerization.