Evidence supporting that the physiologically active configuration of ApoA-I in dHDL is LL5/2

CUELLAR LA; PRIETO ED; CABALEIRO LV; GONZALEZ MC; GARDA HA

Río de Janeiro

Encuentro; III Latin American Posgraduate Program of Biophisics course I colloquium Brazil/Africa of Biophysics; 2010

Latin American Federation of Biophysical Societies / Internal Union of Pure and Applied Biophysics

  Discoidal high-density lipoproteins (dHDL) are key intermediates in apolipoprotein A-I (apoAI) mediated cell lipid efflux. Actual knowledge on these complexes comes from studies using dHDL reconstituted by detergentdialysis[1], although they can also be generated by the spontaneous reaction of apoAI with phospholipid vesicles at their phase transition temperature, a procedure that may be similar to the "in vivo" apoAI lipidation. It is well accepted that dHDL consist of a discoidal lipid bilayer surrounded by two anti-parallel apoAI molecules with their a-helices perpendicular to the lipid hydrocarbon chains. In dHDL obtained by cholate-dialysis, Silva et al. [2] detected two coexistent arragnements: One with helix-5 of each apoAI molecule in juxtaposition (LL5/5), and a second with helix-5 of one apoAI molecule in juxtaposition with helix 2 of the another (LL5/2). To compare the configuration of apoAI in spontaneously generated dHDL with that of dHDL obtained by detergent-dialysis, we measured some intermolecular distances through fluorescence energy transfer using two single tryptophan mutants (W@104 and W@108) and three Alexa-488-labeled cysteine mutants (K107C, K133C and K226C). Data agree with the coexistence of both configurations in dHDL generated by cholate-dialysis, but indicates that only the LL5/2 configuration is present in spontaneously generated dHDL. Moreover, spontaneously generated dHDL are more active than those prepared by detergent-dialysis in promoting cell cholesterol efflux, indicating that only the LL5/2 configuration is functional. As we previously proposed, the central 3-4 helix pairs form an intermolecular membrane-inserting bundle, which is possible in LL5/2 but not in LL5/5 configuration.