CHAPTER 13
The Folding Pathway of Disulfide Containing Proteins
J. Y. CHANG l
1
Summary
The pathway of oxidative folding of four single domain, 3-disulfide containing
proteins has been studied in our laboratory. These four proteins are hirudin core
domain (Hir, 49 amino acids), potato carboxypeptidase inhibitor (PCl, 39 amino
acids), human epidermal growth factor (EGF, 58 amino acids) and tick anticoagulant peptide (TAP, 60 amino acids). Their folding pathways were analyzed by
characterization of the acid and iodoacetate trapped folding intermediates. The
results demonstrate a high degree of heterogeneity of the 1- and 2-disulfide intermediates and the presence of 3-disulfide scrambled species along the folding
pathway of all these four proteins. Their folding mechanism differs significantly
from the well documented case of bovine pancreatic trypsin inhibitor (BPTI).
2
Introduction
The elucidation of "Protein folding pathway" is a major issue and a very much
debated subject in the field of protein chemistry (Baldwin, 1989; Kim & Baldwin,
1990; Creighton, 1990; Richards, 1991; Matthews, 1993). There are limited methodologies available to conduct such analysis. A newly developed technique of
pulsed-label NMR (Roder et ai., 1988; Udgaonkar & Baldwin, 1988; Jennings &
Wright, 1993) permits trapping and identification of amide groups that are
engaged in the structured elements during the process of folding. However, one
of the best established techniques is so called "disulfide folding pathway" pioneered by Creighton (1978 &1990). The disulfide folding pathway is defined by
the kinetics of formation of native disulfide bonds during the process of oxidative
folding (Creighton, 1986). Extensive application of this technique in the past 20
years has produced one major model of protein folding, the disulfide folding
pathway of bovine pancreatic trypsin inhibitor (BPTI) (Creighton, 1990; Weissmann & Kim, 1991), a kunitz-type protease inhibitor which comprises 58 amino
acids and 3 disulfides. Despite the lingering debate about its detailed mechanism
I Research Center for Protein Chemistry, Institute of Molecular Medicine The University of Texas,
Houston, Texas 77030.
The Folding Pathway of Disulfide Containing Proteins
J. Y. CHANG l
1
Summary
The pathway of oxidative folding of four single domain, 3-disulfide containing
proteins has been studied in our laboratory. These four proteins are hirudin core
domain (Hir, 49 amino acids), potato carboxypeptidase inhibitor (PCl, 39 amino
acids), human epidermal growth factor (EGF, 58 amino acids) and tick anticoagulant peptide (TAP, 60 amino acids). Their folding pathways were analyzed by
characterization of the acid and iodoacetate trapped folding intermediates. The
results demonstrate a high degree of heterogeneity of the 1- and 2-disulfide intermediates and the presence of 3-disulfide scrambled species along the folding
pathway of all these four proteins. Their folding mechanism differs significantly
from the well documented case of bovine pancreatic trypsin inhibitor (BPTI).
2
Introduction
The elucidation of "Protein folding pathway" is a major issue and a very much
debated subject in the field of protein chemistry (Baldwin, 1989; Kim & Baldwin,
1990; Creighton, 1990; Richards, 1991; Matthews, 1993). There are limited methodologies available to conduct such analysis. A newly developed technique of
pulsed-label NMR (Roder et ai., 1988; Udgaonkar & Baldwin, 1988; Jennings &
Wright, 1993) permits trapping and identification of amide groups that are
engaged in the structured elements during the process of folding. However, one
of the best established techniques is so called "disulfide folding pathway" pioneered by Creighton (1978 &1990). The disulfide folding pathway is defined by
the kinetics of formation of native disulfide bonds during the process of oxidative
folding (Creighton, 1986). Extensive application of this technique in the past 20
years has produced one major model of protein folding, the disulfide folding
pathway of bovine pancreatic trypsin inhibitor (BPTI) (Creighton, 1990; Weissmann & Kim, 1991), a kunitz-type protease inhibitor which comprises 58 amino
acids and 3 disulfides. Despite the lingering debate about its detailed mechanism
I Research Center for Protein Chemistry, Institute of Molecular Medicine The University of Texas,
Houston, Texas 77030.