A novel folding intermediate state for apolipoprotein A-I: Role of the amino and carboxy termini
Eitan Gross 1*, Dao-Quan Peng 1, Stanley L. Hazen 1 and Jonathan Smith 1
1 Cleveland Clinic Foundation
* To whom correspondence should be addressed. E-mail: eitan.gross{at}case.edu.
Submitted on September 26, 2005
Revised on October 26, 2005
Accepted on 7 November 2005
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Abstract |
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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 which 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 re-folding 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 (Iequil) in addition to the native (N) and unfolded (U) states. Refolding kinetics of apoAI, measured by stopped-flow circular dichroism, revealed two kinetic intermediates, Iburst and Irecovery. Computer modeling suggested the first resembles the partially unfolded protein, while the second overlaps with the native state of the protein. The free energy change for the N --> Iequil transition of the N-terminal and double deletions were lower then that of the full length form, while 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 --> Iequil unfolding transition; while the carboxyl terminus destabilizes that state.
Key Words:
Stopped-flow, circular dichroism, fluorescence spectroscopy
