SOLUTION CONFORMATION OF LEU27 HGRF(1-32)NH2 AND ITS DEAMIDATION PRODUCTS BY 2D NMR

The solution structure and helical content of a human growth hormone r eleasing factor analog, Leu27 hGRF(1-32)NH2 (hGRF), and its deamidatio n products Asp8 Leu27 hGRF(1-32)NH2 and isoAsp8 Leu27 hGRF(1-32)NH2, w ere determined by CD and 2D NMR. Chemical-shift assignments of H-1 NMR resonances were made from DQFCOSY, HOHAHA and NOESY spectra, and qual itative secondary structure was determined from NOESY spectra. 2D NMR studies in aqueous MeOH showed the Asn8, Asp8 and isoAsp8 hGRF analogs to have significant alpha-helical character. However, the beta-linked isoAsp8 analog did not retain helical structure in the N-terminal reg ion, most likely because of disruption of the hydrogen bonding pattern upon substitution of the extra methylene into the peptide backbone. T he helical content, as determined by CD, was approximately 12% in 0% M eOH for all three peptides, and 77, 72 and 69% in 80% MeOH for the Asn 8, Asp8 and isoAsp8 hGRF analogs, respectively. However, 2D NMR soluti on structure data indicated a decrease in helicity in the N-terminal r egion for the isoAsp8 analog when compared with the other two analogs. In the Asn8 and Asp8 hGRF analogs, the helix began at Asp3 or Ala4, w hile the isoAsp8 analog helix was disrupted until Arg11. The higher he licity value for the Asn8 peptide over the isoAsp8 analog may be assoc iated with reported biological activity, where the in vitro activity d ecreased from 100 to 4 and < 1% for Asn8, Asp8 and isoAsp8 hGRF, respe ctively. (C) Munksgaard 1993.