Effect of the intermolecular disulfide bond on the conformation and stability of glial cell line-derived neurotrophic factor.
Glial cell line-derived neurotrophic factor (GDNF) is a member of the TGF-beta superfamily of proteins. It exists as a covalent dimer in solution, with the 15 kDa monomers linked by an interchain disulfide bond through the Cys101 residues. Sedimentation equilibrium and velocity experiments demonstrated that, after removal of the interchain disulfide bond, GDNF remains as a non-covalent dimer and is stable at pH 7.0. To investigate the effect of the intermolecular disulfide on the structure and stability of GDNF, we compared the solution structures of the wild-type protein and a cysteine-101 to alanine (C101A) mutant using Fourier transform infrared (FTIR), FT-Raman and circular dichroism (CD) spectroscopy and sedimentation analysis. The elimination of the intermolecular disulfide bond causes only minor changes (approximately 4%) in the secondary structures of GDNF. The far- and near-UV CD spectra demonstrated that the secondary and tertiary structures were similar for both wild-type and C101A GDNF. Heparin binding and sedimentation velocity experiments also indicated that the folded structure of the wild-type and C101A GDNF are indistinguishable. The thermal stability of GDNF does not appear to be affected by the absence of the interchain disulfide bond and the biological activity of the C101A mutant is identical with that of the wild-type protein. However, small but significant changes in side chain conformations of tyrosine and aliphatic residues were observed by FT-Raman spectroscopy upon removal of the intermolecular disulfide bond, which may reflect structural changes in the area of dimeric contact. By comparing the Raman spectrum of wild-type GDNF with that of the C101A analog, we identified the conformation of the intermolecular disulfide as trans-gauche-trans geometry. These results indicate that GDNF is an active, properly folded molecule in the absence of the interchain disulfide bond.
Li
Tiansheng
T
Pharmaceutics, Amgen Inc., Amgen Center, One Amgen Drive,M/S 8-1-C Thousand Oaks, CA 91320, USA.
Yamane
Harvey
H
Arakawa
Tsutomu
T
Narhi
Linda O
LO
Philo
John
J
D000595:Amino Acid Sequence; D002460:Cell Line; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D051100:Glial Cell Line-Derived Neurotrophic Factor; D006493:Heparin / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000737:chemistry
D004220:Disulfides; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D006493:Heparin; D003545:Cysteine