Structural Studies of N- and C-Terminally Truncated Human Apolipoprotein A-I

Publisher Website
Google Scholar
Abstract
Apolipoprotein A-I (apoA-I) plays an important structural and functional role in lipid transport and metabolism. This work is focused on the central region of apoA-I (residues 60−183) that is predicted to contain exclusively amphipathic α-helices. Six N- and/or C-terminally truncated mutants, Δ(1−41), Δ(1−59), Δ(198−243), Δ(209−243), Δ(1−41,185−243), and Δ(1−59,185−243), were analyzed in their lipid-free state in solution at pH 4.7−7.8 by far- and near-UV CD spectroscopy. At pH 7.8, all mutants show well-defined secondary structures consisting of 40−52% α-helix. Comparison of the α-helix content in the wild type and mutants suggests that deletion of either the N- or C-terminal region induces helical unfolding elsewhere in the structure, indicating that the terminal regions are important for the integrity of the solution conformation of apoA-I. Near-UV CD spectra indicate significant tertiary and/or quaternary structural changes resulting from deletion of the N-terminal 41 residues. Reduction in pH from 7.8 to 4.7 leads to an increase in the mutant helical content by 5−20% and to a large increase in thermal unfolding cooperativity. Van't Hoff analysis of the mutants at pH 4.7 indicates melting temperatures Tm ranging from 51 to 59 °C and effective enthalpies ΔHv(Tm) = 35 ± 5 kcal/mol, similar to the values for plasma apoA-I at pH 7.8 (Tm = 57 °C, ΔHv = 32 kcal/mol). Our results provide the first report of the pH effects on the secondary, tertiary, and/or quaternary structure of apoA-I variants and indicate the importance of the electrostatic interactions for the solution conformation of apoA-I.