Domain-dependent protein folding is indicated by the intracellular kinetics of disulfide bond formation of human chorionic gonadotropin beta subunit.
We have measured the intracellular rates of formation of the six disulfide bonds in the human chorionic gonadotropin beta subunit (hCG-beta) to determine whether the folding pathway of this molecule can be described by a simple sequential model. If such a model is correct, the formation of disulfide bonds, which is indicative of tertiary structural changes during protein folding, should occur in a discrete order. The individual rates of disulfide bridging were determined by identifying the extent of disulfide bond formation in hCG-beta intermediates purified from choriocarcinoma cells that had been metabolically labeled for 40 to 120 s and chased for 0 to 25 min. The results of these kinetic studies describe a folding pathway in which the disulfide bonds between cysteines 34-88, 38-57, 9-90 and 23-72 stabilize, in a discrete order, the putative domain(s) involving amino acids 1-90 of hCG-beta. However, the S-S bonds 93-100 and 26-110 begin to form before the complete formation of the disulfide bonds that stabilize the amino acid 1-90 domain(s), and continue to form after complete formation of these disulfide bonds, suggesting that hCG-beta does not fold by a simple sequential pathway. The order of completion of each of the six disulfide bonds of hCG-beta is: 34-88 (t1/2 = 1-2 min), 38-57 (t1/2 = 2-3 min), 9-90 and 23-72, 93-100, and 26-110. Moreover, 60-100% of each of the six disulfide bonds form posttranslationally, and nonnative disulfide bonds do not form in detectable amounts during intracellular folding of hCG-beta.
Huth
J R
JR
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805.
Mountjoy
K
K
Perini
F
F
Bedows
E
E
Ruddon
R W
RW
D002822:Choriocarcinoma; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D014407:Tumor Cells, Cultured
D006063:Chorionic Gonadotropin; D004220:Disulfides