INIBIOLP   05426
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE LA PLATA "PROF. DR. RODOLFO R. BRENNER"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Studies with pyrenyl-labeled apolipoprotein A I. A multiparametric analysis of Pyrenyl-maleimide.
Autor/es:
FALOMIR LOCKHART LISANDRO; GONZALEZ, MARINA CECILIA; TARRAGA WILSON; GARDA HORACIO ALBERTO
Lugar:
San Luis
Reunión:
Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019
Institución organizadora:
Universidad Nacional de San Luis
Resumen:
Apolipoprotein A‑I (apo A‑I) is the main protein of high-density lipoproteins (HDL), towhich antiatherogenic properties are attributed to its role in the reverse transport ofcholesterol 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 abundle with poorly characterized tertiary and quaternary structures. Depending on theconcentration, apo A‑I self‑aggregates to form dimers and oligomers of higher orders,with a mechanism unwell characterized. It also interacts with phospholipids and formsdiscoidal HDL (dHDL) in different anti‑parallel helical arrangement which differ on theproximity of its helices from each other.The aim of the present study is to obtain information on the apo A‑I self‑aggregation insolution, especially helices proximities, which may be important for understanding themechanisms of dHDL generation.Six cysteine mutants (K107C, K133C F104C, L137C, K226C and F225C) were specificallydesigned and labeled with pyrenyl‑maleimide in positions corresponding to hydrophilicand hydrophobic faces of helices 4, 5 and 10. The monomer and excimer fluorescence ofthe labeled proteins were registered as a function of total apo A‑I concentration; andseveral mathematical models were developed and compared to evaluate the differentassociation types and calculate association constants (Kas) corresponding to thedifferent 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 withphospholipids to form dHDL. Fluorescence emission spectra of pyrene showed excimerformation only in the case of labeled F225C, K133C and K226C mutants, highlighting theparticipation of helices 5 and 10 in the contact regions during certain oligomerizationsteps. Changes in p-value of monomer emission also reported conformational changesduring apo A‑I oligomerization. In the case of K133C, we predicted at least two differentevents of oligomerization, and a model of progressive association seems to be the mostsuitable to represent this behavior.Altogether, these results suggest that the self-proximities of helixes 5 and 10, which arenecessary to form dHDL, are already present in the soluble conformers of apo A-I.