Protein folding and assembly in vitro parallel intracellular folding and assembly: Catalysis of folding and assembly of the human chorionic gonadotropin αβ dimer by protein disulfide isomerase

Jeffrey R. Huth, Fulvio Perini, Oksana Lockridge, Elliott Bedows, Raymond W. Ruddon

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Abstract

Although purified proteins will refold and assemble in vitro, it is not known if cellular factors change the mechanisms of these processes. Based on the gel migration of folding intermediates, the kinetic relationships between these intermediates, and on the order of formation of six disulfide bonds, we have found that the in vitro folding pathway of the human chorionic gonadotropin β subunit (hCG-β) is indistinguishable from the intracellular folding pathway. The same rate-limiting event was found in both folding environments; however, the f1/2 for this step in a cell is 4 min, whereas in vitro the t1/2 was ≥ 80 min. Protein disulfide isomerase (PDI) increased the in vitro rate of this event (t1/2 = 25 min) without changing the order of disulfide bond formation. PDI also catalyzed the in vitro rate of assembly of hCG subunits. In intact cells, assembly of the αβ heterodimer occurs before all of the intramolecular disulfide bonds of β are formed. In vitro, assembly was increased after reduction of two of the carboxylterminal disulfide bonds of hCG-β by PDI. These results strongly suggest that both in intact cells and in vitro, partially unfolded hCG-β is more assembly-competent than is fully folded hCG-β. The comparison of in vitro and intracellular hCG-β folding and hCG subunit assembly which is shown in this report indicates that the assisted folding and assembly pathway that occurs in cells, where proteins such as PDI play a role, differs only in rate but not in the order of disulfide bond formation or in the precursor-product relationships among the folding intermediates.

Original languageEnglish (US)
Pages (from-to)16472-16482
Number of pages11
JournalJournal of Biological Chemistry
Volume268
Issue number22
StatePublished - Aug 5 1993

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Protein Disulfide-Isomerases
Protein folding
Protein Folding
Chorionic Gonadotropin
Catalysis
Dimers
Disulfides
Protein Refolding
In Vitro Techniques
Proteins
Gels
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Protein folding and assembly in vitro parallel intracellular folding and assembly : Catalysis of folding and assembly of the human chorionic gonadotropin αβ dimer by protein disulfide isomerase. / Huth, Jeffrey R.; Perini, Fulvio; Lockridge, Oksana; Bedows, Elliott; Ruddon, Raymond W.

In: Journal of Biological Chemistry, Vol. 268, No. 22, 05.08.1993, p. 16472-16482.

Research output: Contribution to journalArticle

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