Structures of ovine corticotropin-releasing factor and its Ala32 mutant as studied by CD and NMR techniques.
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 alpha-helix formation. In contrast to an earlier report, it was observed the alpha-helical structure extends to the C-terminus of oCRF. By analyzing the CalphaH and NH chemical shifts, the properties of local structures of oCRF were elucidated. The oCRF and [Ala32]oCRF have stable alpha-helical structures in the middle region, regardless of pH 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 substitution region, and it is thought that this is the cause of the increased activity of [Ala32]oCRF.
Ryu
K S
KS
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea.
Choi
B S
BS
Chi
S W
SW
Kim
S H
SH
Kim
H
H
D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003346:Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D012756:Sheep; D013696:Temperature; D014270:Trifluoroethanol
D014270:Trifluoroethanol; D003346:Corticotropin-Releasing Hormone; D000409:Alanine