The Solution Structure of a Cyclic Analog of Neuropeptide Y with High Y 1 Receptor Affinity by NMR, CD and MD Simulations Errol Mathias, 1,4 Arati Prabhu, 2 Evans Coutinho, 2 Sudha Srivastava, 3,5 and Uma Halkar 1 (Accepted September 1, 2004) The conformation of a cyclic analog of neuropeptide Y [Tyr 1 –Lys–Gly–Arg–cyclo 5/8 -(Glu 5 – Tyr–Ile–Lys 8 )–Leu–Ile 10 –Thr–Arg–Pro–Arg–Tyr 15 –NH 2 ; cEK-NPY] with high Y 1 receptor affinity was studied using 1 H, 13 C and 15 N 2D-NMR and CD in three diverse media-viz. DMSO-d 6 , water (pH 4.0) and 50% hexafluoroacetone (HFA). The conformation of cEK- NPY was interpreted based on chemical shift ( 1 H, 13 C and 15 N), temperature coefficients of the NH chemical shifts, 3 J NHa coupling constants and the pattern of intra and inter-residue NOEÕs and the CD spectrum. In both DMSO and water, there is a preponderance of a b-strand structure, while HFA promotes an a-helical structure, which is discontinuous in the mid- region of the peptide, due to the constraints of the lactam ring. The solution structures were generated using Restrained Molecular Dynamics simulations and further refined by Mardigras to R factors between 0.55 and 0.65. The role of its conformations in its biological activity is discussed. KEY WORDS: Molecular dynamics; multinuclear NMR; neuropeptide Y. INTRODUCTION Neuropeptide Y (NPY), a 36 amino acid peptide was isolated from the porcine brain, and subse- quently sequenced in 1982 (Tatemoto et al., 1982; Tatemoto, 1982). NPY is a member of the pancreatic polypeptide family having a high homology with pancreatic polypeptide (PP) and the peptide YY (PYY) (Allen et al., 1987). It is predominantly dis- tributed in the periphery and to some extent in the brain region (Gray and Morley, 1986). Recent spec- tral studies (Allen et al., 1987) and molecular dynamics (MD) simulations, suggest that the tertiary structure of NPY is characterized by a polyproline- type II helix around residues 1–8, a b-turn at posi- tions 9 through 14, an amphipathic a-helical segment extending from residues 15 to 32 and a turn structure for the C-terminal residues 33–36. The earliest and 1 Department of Chemistry, D.G. Ruparel College, Senapati Bapat Marg, Mahim, 400 016, Mumbai, India. 2 Department of Pharmaceutical Chemistry, Bombay College of Pharmacy, Kalina, 400 098, Mumbai, India. 3 Tata Institute of Fundamental Research, Homi Bhabha Road Navy Nagar, Colaba, 400 005, Mumbai, India. 4 Department of Chemistry and Biochemistry, California State University, Los Angeles, CA, 90032-8202, USA. 5 Correspondence should be addressed to: Sudha Srivastava, Tel: +91-22-2280-4545; Fax: +91-22-2280-4682; E-mail: sudha@tifr. res.in. Abbreviations: CD, Circular dichroism; CEK-NPY, Tyr1–Lys– Gly–Arg–cyclo 5/8 -(Glu 5 –Tyr–Ile–Lys 8 )–Leu–Ile 10 –Thr–Arg–Pro– Arg–Tyr 15 –NH 2 ; DMSO, Dimethylsulfoxide; DQF-COSY, Dou- ble quantum filtered correlated spectroscopy; DSS, 2,2¢-Dimethyl- 2-silapentane-5-sulphonic acid; HFA, Hexafluoroacetone; HSQC, Heteronuclear single quantum coherence; MD, Molecular dynamics; NMR, Nuclear magnetic resonance; NPY, Neuropep- tide Y; NOE, Nuclear overhauser effect; NOESY, Nuclear over- hauser enhancement spectroscopy; ROESY, Rotating frame nuclear overhauser effect spectroscopy; SAR, Structure activity relationship; 1D, One-dimensional; 2D, Two-dimensional; TFE, Trifluoroethanol; TOCSY, Total correlation spectroscopy. International Journal of Peptide Research and Therapeutics, Vol. 11, No. 2, June 2005 (Ó 2005), pp. 143–152 DOI: 10.1007/s10989-004-4708-1 143 1573-3149/05/0600–0143/0 Ó 2005 Springer Science+Business Media, Inc.