Structures of ovine corticotropin-releasing factor and its Ala32 mutant asstudied by CD and NMR techniques

The corticotropin-releasing factor (CRF) is a 41-amino acid peptide-amide h ormone, which mediates a general stress-response. It has been reported that the substitution of His-32 in the ovine CRF (oCRF) with Ala brings about a 4.5-fold increase in activity [Kornreich et al. (1992) J. Med. Chem. 35, 1 870-76]. Here, we have determined the secondary structure of this Ala-subst ituted ovine CRF ([Ala32]oCRF) and compare it with that of oCRF using circu lar dichroism (CD) and NMR techniques in trifluoroethanol (TFE) solution, w hich is known to stabilize the alpha-helix formation, In contrast to an ear lier report, it was observed the alpha-helical structure extends to the C-t erminus of oCRF, By analyzing the CalphaH and NH chemical shifts, the prope rties of local structures of oCRF were elucidated. The oCRF and [Ala32]oCRF have stable alpha-helical structures in the middle region, regardless of p H and temperature, and the alpha-helix initiation regions of these peptides are stabilized as the pH is decreased. However, the [Ala32]oCRF has a more stable alpha-helical structure than oCRF in the vicinity of the substituti on region, and it is thought that this is the cause of the increased activi ty of [Ala32]oCRF.