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J. Biol. Chem., Vol. 283, Issue 49, 34393-34402, December 5, 2008

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Effect of Leucine to Phenylalanine Substitution on the Nonpolar Face of a Class A Amphipathic Helical Peptide on Its Interaction with Lipid

HIGH RESOLUTION SOLUTION NMR STUDIES OF 4F-DIMYRISTOYLPHOSPHATIDYLCHOLINE DISCOIDAL COMPLEX^*

Vinod K. Mishra¹, Mayakonda N. Palgunachari, N. Rama Krishna, John Glushka^¶, Jere P. Segrest, and G. M. Anantharamaiah²

From the Atherosclerosis Research Unit, the Department of Medicine, and the Department of Biochemistry and Molecular Genetics and Comprehensive Cancer Center, University of Alabama Medical Center, Birmingham, Alabama 35294 and the ^¶Southeast Collaboratory for Biomolecular NMR/The Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602

Model class A amphipathic helical peptides mimic several properties of apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins. Previously, we reported the NMR structures of Ac-18A-NH₂ (renamed as 2F because of two phenylalanines), the base-line model class A amphipathic helical peptide in the presence of lipid ( Mishra, V. K., Anantharamaiah, G. M., Segrest, J. P., Palgunachari, M. N., Chaddha, M., Simon Sham, S. W., and Krishna, N. R. (2006) J Biol. Chem. 281, 6511-6519[Abstract/Free Full Text] ). Substitution of two Leu residues on the nonpolar face (Leu³ and Leu¹⁴) with Phe residues produced the peptide 4F (so named because of four phenylalanines), which has been extensively studied for its anti-inflammatory and antiatherogenic properties. Like 2F, 4F also forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Since subtle structural changes in the peptide-lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of 4F in 4F·DMPC discs. Like 2F, 4F adopts a well defined amphipathic -helical structure in association with the lipid at a 1:1 peptide/lipid weight ratio. Nuclear Overhauser effect (NOE) spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. Similar to 2F, the pattern of observed peptide-lipid NOEs is consistent with a parallel orientation of the amphipathic helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). However, in contrast to 2F in 2F·DMPC, 4F in the 4F·DMPC complex is located closer to the lipid headgroup as evidenced by a number of NOEs between 4F and DMPC headgroup protons. These NOEs are absent in the 2F·DMPC complex. In addition, the conformation of the DMPC sn-3 chain in 4F·DMPC complex is different than in the 2F·DMPC complex as evidenced by the NOE between lipid 2.CH and βCH₂ protons in 4F·DMPC, but not in 2F·DMPC, complex. Based on the results of this study, we infer that the antiatherogenic properties of 4F may result from its preferential interaction with lipid headgroups.

Received for publication, August 18, 2008 , and in revised form, October 8, 2008.

The atomic coordinates of NMR structures of 4F in 4F-DMPC disc, ¹H chemical shifts, and distance and dihedral constraints (accession number 20034), have been deposited in the Biological Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu).

^* This work was supported, in whole or in part, by National Institutes of Health Grants RO1HL089328, PO1 HL34343 (NHLBI) and CA-13148 (NCI). The 600-MHz CryoProbe was funded by 1S10RR021064-01A1 (NCRR, National Institutes of Health). This research benefited from activities at the SE Collaboratory for High-Field Biomolecular NMR, a research resource at the University of Georgia, funded by NIGMS, National Institutes of Health (NIG MSEC Grant GM66340) and the Georgia Research Alliance. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

² A principal in Bruin Pharma, a startup biotech company.

¹ To whom correspondence should be addressed: D806, 1808 Seventh Ave. South, UAB Medical Center, Birmingham, AL 35294-0012. Tel.: 205-934-4021; Fax: 205-975-8079; E-mail: vmishra{at}uab.edu.

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