ASSIGNMENT OF THE HELICAL STRUCTURE IN NEUROPEPTIDE-Y BY HPLC STUDIESOF METHIONINE REPLACEMENT ANALOGS AND H-1-NMR SPECTROSCOPY

The HPLC retention behavior of three complete single methionine and me thionine sulfoxide replacement sets of two 18-mer model peptides and n europeptide Y (NPY) were investigated. All peptides were prepared by m ultiple solid-phase peptide synthesis. Plotting the retention time dif ferences between methionine and methionine sulfoxide analogues ys the position of replacement shows that potentially alpha-helical peptides become helical on binding during reversed-phase high performance liqui d chromatography. In the case of an amphipathic cy-helix, the retentio n time differences change periodically with a 3-4 repeat pattern, whic h allow the location of amphipathic helical structures. Replacements i n nonamphipathic alpha-helical domains cause focal preferential bindin g areas and lead to sequence-dependent retention time profiles. Methio nine replacement studies of NPY suggest an unstructured or extended co nformation from Tyr(1) to Ala(12) connected to a well-defined amphipat hic alpha-helix from Pro(13) to Arg(35). The assignment is confirmed b y comparison of nuclear Overhauser effects based two-dimensional H-1-n mr spectroscopy and utilization of the C-alpha H shift index method in 50% trifluoroethanol/50% water. (C) 1996 John Wiley & Sons, Inc.