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

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

Salta

Congreso; XXXIX Annual Meeting of the Argentinean Biophysical Society / 3rd Latin American Protein Society Meeting; 2010

Sociedad Argentina de Biofísica / Latin American Protein Society

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> 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 detergent-dialysis1, 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. 1. Matz, C. E. and A. Jonas, J Biol Chem. Micellar complexes of human apolipoprotein A-I with phosphatidylcholine and cholesterol prepared from cholate-lipid dispersions. 257(8): p. 4535-40 (1982) 2. Silva, R. A. et al., Biochemistry. A mass spectroscopic determination of the conformation of dimeric apolipoprotein A-I in discoidal high density lipoproteins. 44(24): p. 8600-7 (2005)   Aknowledgements: Hernandez, Laura for technical support. CONICET, ANPCyT and UNLP for financial support