Effect of secondary structure on the rate of deamidation of several growth hormone releasing factor analogs.

Abstract

The objective of this study was to determine whether the rates of deamidation of Asn8 in selected growth hormone releasing factor (GRF) analogs were related to the peptide's secondary structures in solution. Bovine or human [Leu27]GRF(1-32)NH2 (both having Gly at position 15), [Ala15Leu27]bGRF(1-32)NH2 and [Pro15Leu27]bGRF(1-32)NH2 were used as model peptides. The peptide helical content (assessed by CD) increased with the increasing methanol concentration and was as follows: 7, 12 and 18% in 0% MeOH; 24, 48 and 52% in 40% MeOH; and 41, 77 and 81% in 80% MeOH for Pro15Leu27 bGRF(1-32)NH2, [Leu27]hGRF(1-32)NH2 and Ala15Leu27 bGRF(1-32)NH2, respectively. 2D NMR studies done in the presence of 40% CD3OH indicated more helical structure for the Ala15 analog as compared to [Leu27]hGRF(1-32)NH2. In both these peptides Asn8 was included in the helical region. In contrast, the lack of conformational information for the Pro15 analog indicated little helical structure around Asn8. The peptides' deamidation rates decreased and their half-lives increased with increasing MeOH concentrations. At 40% MeOH, the least helical Pro15 bGRF analog (t1/2 = 10.78 h) deamidated 1.5 and 2 times faster than its Gly15 (t1/2 = 15.74 h) and Ala15 (t1/2 = 21.53 h) counterparts, respectively. This study indicates that helical environment around Asn8 in GRF makes this residue less prone to deamidation.