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

Abstract

The corticotropin-releasing factor (CRF) is a 41-amino acid peptide- amide hormone, 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, 1870-76]. Here, we have determined the secondary structure of this Ala- substituted ovine CRF ([Ala32]oCRF) and compare it with that of oCRF using circular dichroism (CD) and NMR techniques in trifluoroethanol (TFE) solution, which is known to stabilize the α-helix formation. In contrast to an earlier report, it was observed the α-helical structure extends to the C- terminus of oCRF. By analyzing the C(α) and NH chemical shifts, the properties of local structures of oCRF were elucidated. The oCRF and [Ala32]oCRF have stable α-helical structures in the middle region, regardless of pH and temperature, and the α-helix initiation regions of these peptides are stabilized as the pH is decreased. However, the [Ala32]oCRF has a more stable α-helical structure than oCRF in the vicinity of the substitution region, and it is thought that this is the cause of the increased activity of [Ala32]oCRF.