The role of disulfide bonds in the structure and function of murine epidermal growth factor (mEGF).
A systematic study using solid phase peptide synthesis has been undertaken to examine the role of the disulfide bonds in the structure and function of mEGF. A combination of one, two and three native disulfide pair analogues of an active truncated (4-48) form of mEGF have been synthesised by replacing specific cysteine residues with isosteric a-amino-n-butyric acid (Abu). Oxidation of the peptides was performed using either conventional aerobic oxidation at basic pH, in DMSO under acidic conditions or via selective disulfide formation using orthogonal protection of the cysteine pairs. The contribution of individual, or pairs of, disulfide bonds to EGF structure was evaluated by CD and (1)H-NMR spectroscopy. The mitogenic activity of each analogue was determined using Balb/c 3T3 mouse fibroblastsAs we have reported previously (Barnham et al. 1998), the disulfide bond between residues 6 and 20 can be removed with significant retention of biological activity (EC50 20-50 nM). The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. We now show that removal of any other disulfide bond, either singly or in pairs, results in a major disruption of the tertiary structure, and a large loss of activity (EC50>900 nM). Remarkably, the linear analogue appears to have greater activity (EC50 580 nM) than most one and two disulfide bond analogues although it does not have a definable tertiary structure.
Alewood
Dianne
D
The Institute for Molecular Bioscience, The University of Queensland, Australia.
Nielsen
Kathy
K
Alewood
Paul F
PF
Craik
David J
DJ
Andrews
Peter
P
Nerrie
Maureen
M
White
Sara
S
Domagala
Teresa
T
Walker
Francesca
F
Rothacker
Julie
J
Burgess
Antony W
AW
Nice
Edouard C
EC
D000595:Amino Acid Sequence; D000613:Aminobutyrates / Q000494:pharmacology; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D000613:Aminobutyrates; D004220:Disulfides; D010455:Peptides; D004815:Epidermal Growth Factor; D003545:Cysteine; D010100:Oxygen