STRUCTURAL-ANALYSIS OF APOLIPOPROTEIN-A-I - EFFECTS OF AMINO-TERMINALAND CARBOXY-TERMINAL DELETIONS ON THE LIPID-FREE STRUCTURE

An amino-terminal deletion mutant (residues 1-43) and a carboxy-termin al deletion mutant (residues 187-243) of human apolipoprotein A-I (ape hA-I) have been produced from a bacterial expression system to explor e the importance of the missing residues for the conformation of apo h A-I. Our focus has been to study the lipid-free structure of apo hA-I to understand how discrete domains influence the conformational plasti city of the protein and, by inference, the mechanism of lipid binding. All spectral and physical measurements indicate that both apo Delta(1 -43)A-I and apo Delta(187-243)A-I have folded, tertiary structures. Th ese structures differ in the specific arrangement of helical domains b ased, in part, on their relative thermodynamic stability, near-and far -UV CD, limited proteolysis, and the accessibility of tryptophans to f luorescence quenchers. In addition, all data indicate that the folded domains of apo hA-I and apo Delta(187-243)A-I are very similar. Result s from analytical ultracentrifugation suggest that lipid-free apo hA-I and the deletion mutants each exist in a dynamic equilibrium between a loosely folded, helical bundle and an elongated monomeric helical ha irpin. The conformational heterogeneity is consistent with significant ANS binding exhibited by all three proteins and could help to explain the facile lipid binding properties of apo hA-I.