Pyrenyl-maleimide. A multiparametric fluorescence probe. Studies with cysteine mutants of human apolipoprotein A-I

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

La Plata

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

INIBIOLP-UNLP

Apolipoprotein A-I (apo A-I) is the major protein of high-density lipoproteins (HDL), to which antiatherogenic properties are attributed to 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, 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. And the monomer and excimer fluorescence of these labeled proteins were evaluated with different mathematical models to calculate different dissociation constants (Kds).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, K133C and K226C mutants, indicating the participation of helices 5 and 10 in the contact regions for oligomerization.