CONICET | Buscador de Institutos y Recursos Humanos

Discoidal high density lipoproteins (dHDL) are very important intermediates in cell lipid efflux mediated by apolipoprotein A-I (apoAI). There is a general agreement that dHDL consist of a discoidal lipid bilayer surrounded by a belt of two anti-parallel apoAI molecules with most of their a-helices extended and with their long axis perpendicular to the lipid hydrocarbon chains. Most of the structural data has been obtained in dHDL reconstituted by detergent-dialysis, supporting the belt model with a variable helix registry. A major configuration with helix 5 of each apoAI molecule lying in direct opposition (5/5 configuration) was detected, accompanied by minor proportion of 5/2 configuration.dHDL can also be generated by the spontaneous reaction of apoAI with phospholipid vesicles at their phase transition temperature, a procedure that can be similar to the "in vivo" apoAI lipidation. The comparison of the fluorescence energy homotransfer efficiency in dHDL made by both methods with two single tryptophan mutants (W@104 and W@108), suggested that the 5/2 configuration should be the preferred one in the spontaneously generated dHDL.Here we have constructed three cystein apoAI mutants (K107C, K133C and K226C) and labeled them with Alexa-Fluor 488 to get more detailed information on the apoAI configuration in spontaneously generated dHDL. The distances obtained from the efficiency of resonance energy homotransfer (£ 1.5 nm for positions 107 and 226, and 2.5-2.8 nm for position 133) are totally compatible with the 5/2 double belt configuration. The 5/2 registry, in opposition to the 5/5 one, allows a close proximity of the central pair of type Y a-helices and can explain several of the observed properties as well as the interaction of this apoAI region with lipid membranes.