Neuropeptide Y. Optimized solid-phase synthesis and conformational analysis in trifluoroethanol.
The 36-amino-acid neuropeptide Y (human), which is one of the most potent vasoconstrictors and which exhibits a number of other biological functions, has been synthesized using automated peptide synthesis. The optimized method, using 9-fluorenylmethoxycarbonyl protecting and single-step coupling, yielded the crude product in 90% purity allowing for single-step reversed-phase HPLC purification to greater than 98% purity and a high overall yield (50%). The hormone was characterized by several chromatographic methods, ion-spray mass spectroscopy and Edman degradation. The conformation of human neuropeptide Y was examined by CD, NMR and computer simulations. The CD measurements in trifluoroethanol/water (9:1) show a large percentage of alpha-helix. Variation of concentration, from 0.5 microM increasing up to the 1 mM used for NMR measurements, indicates no evidence for aggregation. In the same solvent system, the NMR line widths were very broad and therefore the resonance assignment was achieved with the exclusive use of two-dimensional NOE spectra. The 248 clearly distinguishable NOEs from the NMR study were used in distance geometry calculations and the resulting structures were refined with restrained molecular dynamics. The results indicate an alpha-helix extending from Arg19 to Gln34. For the N-terminal half of the molecule no regular structure was observed.
Mierke
D F
DF
Organisch Chemisches Institut, Technische Universität München, Federal Republic of Germany.
Dürr
H
H
Kessler
H
H
Jung
G
G
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007202:Indicators and Reagents; D007668:Kidney / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter / Q000378:metabolism; D014270:Trifluoroethanol
D007202:Indicators and Reagents; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D017981:Receptors, Neurotransmitter; D014270:Trifluoroethanol