Solution conformation of Leu27 hGRF(1‐32)NH2 and its deamidation products by 2D NMR

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Abstract
The solution structure and helical content of a human growth hormone releasing factor analog, Leu27 hGRF(1–32)NH2 (hGRF), and its deamidation products Asp8 Leu27 hGRF(l-32)NH2 and isoAsp8 Leu27 hGRF(1-32)NH2, were determined by CD and 2D NMR. Chemical-shift assignments of 1H NMR resonances were made from DQFCOSY, HOHAHA and NOESY spectra, and qualitative secondary structure was determined from NOESY spectra. 2D NMR studies in aqueous MeOH showed the Asn8, Asp8 and isoAsp8 hGRF analogs to have significant α-helical character. However, the β-linked isoAsp8 analog did not retain helical structure in the N-terminal region, most likely because of disruption of the hydrogen bonding pattern upon substitution of the extra methylene into the peptide backbone. The helical content, as determined by CD, was ~ 12% in 0% MeOH for all three peptides, and 77, 72 and 69% in 80% MeOH for the Asn8, Asp8 and isoAsp8 hGRF analogs, respectively. However, 2D NMR solution structure data indicated a decrease in helicity in the N-terminal region 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, while the isoAsp8 analog helix was disrupted until Arg. The higher helicity value for the Asn8 peptide over the isoAsp8 analog may be associated with reported biological activity, where the in vitro activity decreased from 100 to 4 and < 1% for Asn8, Asp8 and isoAsp8 hGRF, respectively.

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