Abstract
Apolipoprotein (apo) A-I is thought to undergo a conformational change during lipid association that results in the transition of random coil to alpha-helix. Using a series of deletion mutants lacking different regions along the molecule, we examined the contribution of alpha-helix formation in apoA-I to the binding to egg phosphatidylcholine (PC) small unilamellar vesicles (SUV). Binding isotherms determined by gel filtration showed that apoA-I binds to SUV with high affinity and deletions in the C-terminal region markedly decrease the affinity. Circular dichroism measurements demonstrated that binding to SUV led to an increase in alpha-helix content, but the helix content was somewhat less than in reconstituted discoidal PC.apoA-I complexes for all apoA-I variants, suggesting that the helical structure of apoA-I on SUV is different from that in discs. Isothermal titration calorimetry showed that the binding of apoA-I to SUV is accompanied by a large exothermic heat and deletions in the C-terminal regions greatly decrease the heat. Analysis of the rate of release of heat on binding, as well as the kinetics of quenching of tryptophan fluorescence by brominated PC, indicated that the opening of the N-terminal helix bundle is a rate-limiting step in apoA-I binding to the SUV surface. Significantly, the correlation of thermodynamic parameters of binding with the increase in the number of helical residues revealed that the contribution of alpha-helix formation upon lipid binding to the enthalpy and the free energy of the binding of apoA-I is -1.1 and -0.04 kcal/mol per residue, respectively. These results indicate that alpha-helix formation, especially in the C-terminal regions, provides the energetic source for high affinity binding of apoA-I to lipids.
Highlights
The 243-amino-acid polypeptide chain of apoA-I is organized into characteristic 22- and 11-residue tandem repeats that are predicted to form amphipathic ␣-helices [8]
Based on spectroscopic and calorimetric measurements, we have recently proposed a twostep mechanism for the lipid binding of apoA-I: apoA-I initially binds to a lipid surface through ␣-helices in the C-terminal domain followed by the opening of the helix bundle in the N-terminal domain [13]
We further examined the conformational change of apoA-I upon lipid binding on the small unilamellar vesicles (SUV) surface
Summary
Materials—Egg PC was purchased from Sigma. 1-Palmitoyl,2-oleoyl PC (POPC) was from Avanti Polar Lipids (Pelham, AL). 1-Palmitoyl-2stearoyl-(6-7)-dibromo-phosphatidylcholine (6,7-diBrPC) was from Molecular Probes (Eugene, OR). 1,2-[3H]Cholesterol and [14C]formaldehyde were purchased from PerkinElmer Life Sciences. For gel filtration experiments requiring incorporation of radioactive lipid, [3H]cholesterol in trace amounts (Ͻ1 mol %) was dried along with egg PC in a glass tube and processed as described above for unlabeled SUV. The apoA-I variants were radiolabeled to a specific activity of ϳ1 Ci/mg of protein by reductive methylation of lysine residues with [14C]formaldehyde as described [37, 38]. Fresh SUV (1 mg/ml egg PC) containing a trace amount of [3H]cholesterol was incubated with shaking for 1 h at room temperature with increasing concentrations (10 –100 g/ml) of 14C-labeled apoA-I. After dialyzing from 6 M guanidine hydrochloride solution, the apoA-I sample was diluted to 25 g/ml in 10 mM sodium phosphate buffer (pH 7.4), and the CD spectrum was obtained. The results were corrected by subtracting the buffer baseline or a blank
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
