Mutational effects on the spectroscopic properties and biological activities of oxidized bovine adrenodoxin, and their structural implications.

Of the aromatic 1H-NMR signals of oxidized bovine adrenodoxin only those of His56 showed intrinsic chemical shift changes upon replacement of Tyr82 by Ser or Leu, that must arise from a loss of a through-space ring-current effect of the tyrosine ring in these mutants. Thus, of the three His residues contained in adrenodoxin, His56 is closest to Tyr82, and hence to the highly acidic determinant region of adrenodoxin that is the interaction site for adrenodoxin reductase and P-450. The strong dependence of the fluorescence intensity of Tyr82 on the residue in position 56 supported this observation. As a consequence of this, the effects of replacement of His56 by Gln or Thr on cytochrome c reduction and cytochromes P-450(11 beta) (CYP11B1)-dependent and P-450scc (CYP11A1)-dependent substrate conversions were studied. No influence on Vmax values was observed for all reactions mediated by the mutants, implying His56 does not play a decisive role in the intramolecular or intermolecular electron transfer. In contrast, the Km values were increased, as was the Ks value for binding of CYP11A1 to the [H56T]adrenodoxin. The secondary structure deduced from further NMR data of adrenodoxin was compared with that of other ferredoxins. Tyr82 is in a region of the molecule containing no secondary-structure elements. The data for Tyr82 are in keeping with the biological activities and suggests it is in a flexible, solvent-exposed region of the molecule.

Synthesis, solution structure and biological action of PACAP-related peptide.

High quality PACAP-related peptide (PRP), a 29 amino-acid region of the PACAP precursor protein, has been synthesized in quantities sufficient for biological and structural studies. PRP has a distinct biological activity on the gallbladder that is similar to PACAP, but opposite to that of VIP and its related peptide, PHM. Its solution structure has been investigated by circular dichroism spectroscopy and 2D 1H nuclear magnetic resonance spectroscopy. In contrast to the poorly defined structure in aqueous solution alone, the limiting structure, under conditions that mimic a membrane-like environment, possesses stable secondary structure with a helical region between residues 3 and 20, that is terminated by the presence of glycine at residue 21 and is followed by a region of nascent helix. The similarities and differences in the structure of PRP, PACAP27 and GHRH(1-29) are made through comparison of their H alpha chemical shift data and differences in their biological activities assessed.

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

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.