A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways.
The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.
Wilkins
J A
JA
Department of Protein Chemistry, Otsuka America Pharmaceutical Co., Rockville, Maryland 20850.
Cone
J
J
Randhawa
Z I
ZI
Wood
D
D
Warren
M K
MK
Witkowska
H E
HE
D000595:Amino Acid Sequence; D003545:Cysteine; D004220:Disulfides; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
D004220:Disulfides; D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor; D003545:Cysteine