Conformational analysis of intermediates involved in the in vitro folding pathways of recombinant human macrophage colony stimulating factor beta by sulfhydryl group trapping and hydrogen/deuterium pulsed labeling.
In vitro oxidative folding of reduced recombinant human macrophage colony stimulating factor beta (rhm-CSFbeta) involves two major events: disulfide isomerization in the monomeric intermediates and disulfide-mediated dimerization. Kinetic analysis of rhm-CSFbeta folding indicated that monomer isomerization is slower than dimerization and is, in fact, the rate-determining step. A time-dependent determination of the number of free cysteines remaining was made after refolding commence. The folding intermediates revealed that rhm-CSFbeta folds systematically, forming disulfide bonds via multiple pathways. Mass spectrometric evidence indicates that native as well as non-native intrasubunit disulfide bonds form in monomeric intermediates. Initial dimerization is assumed to involve formation of disulfide bonds, Cys 157/159-Cys' 157/159. Among six intrasubunit disulfide bonds, Cys 48-Cys 139 and Cys' 48-Cys' 139 are assumed to be the last to form, while Cys 31-Cys' 31 is the last intersubunit disulfide bond that forms. Conformational properties of the folding intermediates were probed by H/D exchange pulsed labeling, which showed the coexistence of noncompact dimeric and monomeric species at early stages of folding. As renaturation progresses, the noncompact dimer undergoes significant structural rearrangement, forming a native-like dimer while the monomer maintains a noncompact conformation.
Zhang
Y Heidi
YH
Department of Chemistry, Oregon State University, Corvallis, OR 97330, USA.
Yan
Xuguang
X
Maier
Claudia S
CS
Schimerlik
Michael I
MI
Deinzer
Max L
ML
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry*; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D004220:Disulfides; D011522:Protons; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D016173:Macrophage Colony-Stimulating Factor; D003903:Deuterium; D003545:Cysteine