Analysis of pyrene-labelled apolipoprotein A-I oligomerization in solution: Spectra deconvolution and changes in P-value and excimer formation

TÁRRAGA, WILSON A.; FALOMIR-LOCKHART, LISANDRO J.; GARDA, HORACIO A.; GONZÁLEZ, MARINA C.

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

ELSEVIER SCIENCE INC

Año: 2021

0003-9861

ApoA-I is the main protein of HDL which has anti-atherogenic properties attributed toreverse cholesterol transport. It shares with other exchangeable apolipoproteins a highlevel of structural plasticity. In the lipid-free state, the apolipoprotein amphipathicα-helices interact intra- and inter-molecularly, providing structural stabilization by acomplex self-association mechanism. In this study, we employed a multi-parametricfluorescent probe to study the self-association of apoA-I. We constructed six singlecysteine mutants spanning positions along three helices: F104C, K107C (H4), K133C,L137C (H5), F225C and K226C (H10); and labelled them with N-Maleimide Pyrene.Taking advantage of its spectral properties, namely formation of an excited dimer(excimer) and polarity-dependent changes in its fluorescence fine structure (P-value),we monitored the apoA-I self-association in its lipid-free form as a function of itsconcentration. Interactions in helices H5 (K133C) and H10 (F225C and K226C) werehighlighted by excimer emission; while polarity changes were reported in helix H4(K107C), as well as in helices H5 and H10. Mathematical models were developed toenrich data analysis and estimate association constants (KA) and oligomeric speciesdistribution. Furthermore, we briefly discuss the usefulness of the multi-parametricfluorescent probe to monitor different equilibria, even at a single labelling position.Results suggest that apoA-I self-association must be considered to fully understand itsphysiological roles. Particularly, some contacts that stabilize discoidal HDL particlesseem to be already present in the lipid-free apoA-I oligomers.