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

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), [Ala¹⁵ Leu²⁷]bGRF(1–32)NH₂ and [Pro¹⁵ Leu²⁷]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 Pro¹⁵ Leu²⁷ bGRF(1–32)NH₂, [Leu²⁷]hGRF(1–32)NH₂, and Ala¹⁵ Leu²⁷ 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 [Len²⁷]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 (t_1/2= 10.78 h) deamidated 1.5 and 2 times faster than its Gly¹⁵ (t_1/2= 15.74 h) and Ala¹⁵ (t_1/2= 21.53 h) counterparts, respectively. This study indicates that helical environment around Asn⁸ in GRF makes this residue less prone to deamidation.