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A Novel Folding Intermediate State for Apolipoprotein A-I: Role of the Amino and Carboxy Termini

Eitan Gross ^*, Dao-Quan Peng ^*, Stanley L. Hazen ^*    and Jonathan D. Smith ^*  

^* Department of Cell Biology, Department of Cardiovascular Medicine, and Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic Foundation, Cleveland, Ohio; and Department of Molecular Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio

Correspondence: Address reprint requests to Eitan Gross, PhD, Dept. of Cell Biology, NC-10 Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-844-6333; Fax: 216-844-6339; E-mail: eitan.gross{at}case.edu.

Intramolecular interactions between the amino and carboxy termini of apolipoprotein A-I (apoAI) are believed to stabilize the helix bundle conformation of the protein. During lipid assembly the protein undergoes conformational changes that result in an exposure of the carboxy terminus and its insertion into the lipid phase. To determine the role of the two termini in the energetics of unfolding, we studied the guanidine-hydrochloride-induced unfolding and refolding of apoAI as well as its N-terminal deletion (del[1–43]), C-terminal deletion (del[186–243]), and the double deletion containing only the central residues 44–185. Thermodynamic analysis of the equilibrium unfolding measured by fluorescence spectroscopy revealed the presence of an intermediate unfolded state (I_equil) in addition to the native (N) and unfolded states. Refolding kinetics of apoAI, measured by stopped-flow circular dichroism, revealed two kinetic intermediates, I_burst and I_recovery. Computer modeling suggested that the first resembles the partially unfolded protein, whereas the second overlaps with the native state of the protein. The free energy changes for the N I_equil transition of the N-terminal and double deletions were lower then that of the full-length form, whereas that for the C-terminal deletion was higher. Our findings suggest that the N-terminus of apoAI stabilizes the native state of the protein by increasing the Eyring energy barrier for the N I_equil unfolding transition; whereas the carboxyl terminus destabilizes that state.

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