Solution NMR structure of a model class A (apolipoprotein) amphipathic  helical peptide Vinod K. Mishra a, *, Mayakonda N. Palgunachari a , G.M. Anantharamaiah a,b , Martin K. Jones a , Jere P. Segrest a,b , N. Rama Krishna b, * a The Atherosclerosis Research Unit, Department of Medicine, Birmingham, AL 35294, USA b Department of Biochemistry and Molecular Genetics and Comprehensive Cancer Center, UAB Medical Center, Birmingham, AL 35294, USA Received 9 June 2000; accepted 9 November 2000 Abstract To better understand the structural determinants of the physical-chemical and the biological properties of Ac-18A-NH 2 (acetyl- AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe-amide), we have determined its structure in 50% (v/v) trifluroethanol (TFE-d 3 )/water mixture (5 mM potassium phosphate, pH 5.5, 310K) using two-dimensional proton NMR spectroscopy. Stereospecific assignments have been made for C  H protons (all the residues except Ala and Val) and CH 3 (Val) groups. Nuclear Overhauser effects are observed between the nonpolar side chains spaced at (i) and (i + 4) position in the primary sequence, e.g., Trp2 and Phe6, and Phe6 and Val10. This suggests that in addition to N-terminal acetyl and C-terminal amide groups, the amphipathic  helical structure of Ac-18A-NH 2 is further stabilized by interactions between the hydrophobic residues on the nonpolar face of the helix. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Amphipathic  helix; Peptide; NMR; Apolipoproteins; Trifluoroethanol 1. Introduction The amphipathic  helix (henceforth referred to as am- phipathic helix) is an often encountered secondary structural motif in lipid-associating peptides and proteins [2,31–33]. An amphipathic helix is characterized by a distinct segre- gation of polar and nonpolar amino acid residues along the length of the helix. Amphipathic helices present in ex- changeable apolipoproteins possess positively charged amino acid residues at the polar and nonpolar interface and negatively charged amino acid residues at the center of the polar face [32]. In the original classification of amphipathic helices, such amphipathic helices were grouped into class A (apolipoprotein class) [31]. A number of studies have pro- vided strong experimental evidence for the involvement of amphipathic  helices in the lipid-association of apolipopro- teins, as was postulated by one of us several years ago [30]. Apolipoproteins not only function to stabilize apolar surfaces of lipoprotein particles but also serve as ligands for lipoprotein receptors on cell surfaces [33]. There is a great deal of interest in understanding the structure-function re- lationships of apolipoproteins because of their direct in- volvement in many diseases, especially cardiovascular dis- ease [27]. Over the past several years, our laboratory has been involved in the design and studies of relatively small synthetic apolipoprotein-mimic peptides. An 18 residue peptide, namely 18A, was designed to mimic the distribu- tion of charged amino acid residues in the polar face of the amphipathic helices present in exchangeable apolipopro- teins [1,7]. The peptide 18A has the following amino acid sequence: AspTrpLeuLysAlaPheTyrAspLysValAlaGluLys LeuLysGluAlaPhe. It has been shown that acetylating the N-terminal end and amidating the C-terminal end of 18A to produce Ac-18A-NH 2 results in helix stabilization and in- Abbreviations: CSI, chemical shift index; DQF-COSY, double quan- tum filtered correlation spectroscopy; DSS, sodium 2,2-dimethyl-2-silap- entane-5-sulfonate; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; TFE-d 3 , 2,2,2-trifluoro- ethanol perdeuterated; TOCSY, total correlation spectroscopy; r.m.s.d., root mean square deviation; TPPI, time proportional phase incrementation. * Corresponding author. Tel.: +1-205-934-4023; fax: +1-205-975- 8079. E-mail addresses: vmishra@uab.edu (V.K. Mishra), NRKrishna@ bmg.bhs.uab.edu (N.R. Krishna). 1 Coordinates of all the 25 accepted and the energy minimized average NMR structures of Ac-18A-NH 2 are available upon request. Peptides 22 (2001) 567–573 0196-9781/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S0196-9781(01)00365-5