Structural studies of N- and C-terminally truncated human apolipoprotein A-I.
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 alpha-helices. Six N- and/or C-terminally truncated mutants, delta(1-41), delta(1-59), delta(198-243), delta(209-243), delta(1-41,185-243), and delta(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% alpha-helix. Comparison of the alpha-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 T(m) ranging from 51 to 59 degrees C and effective enthalpies deltaH(v)(T(m)) = 35 +/- 5 kcal/mol, similar to the values for plasma apoA-I at pH 7.8 (T(m) = 57 degrees C, deltaH(v) = 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.
Fang
Yiling
Y
Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, Massachusetts 02118, USA.
Gursky
Olga
O
Atkinson
David
D
D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008325:Mammary Neoplasms, Experimental; D051379:Mice; D008958:Models, Molecular; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D055672:Static Electricity; D013816:Thermodynamics; D014407:Tumor Cells, Cultured
D016632:Apolipoprotein A-I; D010446:Peptide Fragments