Solution structure of pituitary adenylate cyclase activating polypeptide by nuclear magnetic resonance spectroscopy.

Abstract

The solution structures of the recently discovered neuropeptides PACAP38 and PACAP27 have been investigated in aqueous solution containing varying amounts of trifluoroethanol (TFE) by circular dichroism (CD) spectroscopy and a combination of 2D 1H nuclear magnetic resonance (NMR) spectroscopy, distance geometry, and refined molecular dynamics and energy minimization calculations. In aqueous solution both peptides show only small transitory amounts of stable structure while in 50% TFE they adopt ordered structures. Qualitative NOE data and the use of the chemical shift index of the alpha-protons identified the positions of alpha-helical regions. A set of low-energy conformations compatible with the quantitative NOE data were obtained for both and each set were subjected to RMS analysis to determine the positions of the secondary structure elements. PACAP38 has an initial disordered N-terminal domain of eight amino acids, followed by an alpha-helical structure stretching from Ser-9 to Val-26, which contains a discontinuity between Lys-20 and Lys-21, and in the C-terminal region there is a short alpha-helix between Gly-28 and Arg-34. The structure of PACAP27 mirrors remarkably closely that of PACAP38 and shows no fraying of the C-terminal helix. The physiological significance of the three structural domains (1-8, 9-26, and 27-38) of PACAP38 is shown by a comprehensive review of recent in vitro and in vivo investigations of PACAP analogues. The correspondence of the global structural features of PACAP with other members of this family of peptides (namely, secretin, glucagon, GHRF1-29 and VIP) is demonstrated by inspection of the chemical shift indices of the alpha-protons.