Assignment of the helical structure in neuropeptide Y by HPLC studies of methionine replacement analogues and 1H-NMR spectroscopy.
The HPLC retention behavior of three complete single methionine and methionine sulfoxide replacement sets of two 18-mer model peptides and neuropeptide Y (NPY) were investigated. All peptides were prepared by multiple solid-phase peptide synthesis. Plotting the retention time differences between methionine and methionine sulfoxide analogues vs the position of replacement shows that potentially alpha-helical peptides become helical on binding during reversed-phase high performance liquid chromatography. In the case of an amphipathic alpha-helix, the retention time differences change periodically with a 3-4 repeat pattern, which allow the location of amphipathic helical structures. Replacements in nonamphipathic alpha-helical domains cause local preferential binding areas and lead to sequence-dependent retention time profiles. Methionine replacement studies of NPY suggest an unstructured or extended conformation from Tyr1 to Ala12 connected to a well-defined amphipathic alpha-helix from Pro13 to Arg35. The assignment is confirmed by comparison of nuclear Overhauser effects based two-dimensional 1H-nmr spectroscopy and utilization of the C alpha H shift index method in 50% trifluoroethanol/50% water.
Rothemund
S
S
Institute of Molecular Pharmacology, Berlin, Germany.
Krause
E
E
Beyermann
M
M
Bienert
M
M
Sykes
B D
BD
Sönnichsen
F D
FD
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D009478:Neuropeptide Y / Q000031:analogs & derivatives* / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011522:Protons
D009478:Neuropeptide Y; D011522:Protons; D008715:Methionine