Self-aggregation of human apolipoprotein A-I. Studies with pyrenyl-labeled cysteine mutants

TÁRRAGA WILSON; JUAN D. TOLEDO; FALOMIR LISANDRO; GONZALEZ MARINA CECILIA; GARDA, H. A.

Tucumán

Congreso; III Latin American Federation of Biophysical Societies (LAFeBS) | IX Ibero American Congress of Biophysics, XLV Reunión Anual de la Sociedad argentina de Biofísica (SAB); 2016

Sociedad Argentina de Biofísica

Apolipoprotein A-I (apo A-I) is the major protein of high density lipoproteins (HDL), to which antiatherogenic properties are attributed for its role in the 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 defined tertiary structure. Depending on the concentration, apo A-I is self-aggregated to form dimers and oligomers of different size. It is also interacts with membrane and forms discoidal HDL (dHDL).The aim was to obtain information on the apo A-I self-aggregation in solution, as well as its interaction with membrane and formation of complex with lipid, since it is important to understand the mechanisms of HDL generation. Six cysteine mutants (K107C, K133C F104C, L137C, K226C and F225C) were specifically labeled with pyrenylmaleimide in these six positions corresponding to the hydrophilic and hydrophobic faces of helices 4, 5 and 10.The labeled mutants were stable in solution as indicated by tryptophan fluorescence. With the exception of F104C, they were biologically active since they interact with lipids to form dHDL. Fluorescence emission spectra of pyrene in function of the protein concentration showed that pyrene excimer formation occurs only in the case of labeled F225C and K133C mutants, indicating the participation of helices 5 and 10 in the contact regions for oligomerization. Excimer fluorescence was undetectable for all the mutants in the presence of lipid vesicles.