Oxidative Protein Folding in vitro: a
Study of the Cooperation between
Quiescin-sulfhydryl Oxidase and
Protein Disulfide Isomerase
Pumtiwitt C. Rancy and Colin Thorpe*
Department of Chemistry and Biochemistry, University of Delaware
Lingxi Jiang
Oxidative protein folding
• A process that is responsible for the formation of disulfide bonds between cysteine residues in proteins.
• Driving force: a redox reaction, in which electrons are passed between several proteins and finally to a terminal electron acceptor.
2 RSH + O2 → RS-SR + H2O2
• In eukaryotes, the process of oxidative protein folding occurs in the endoplasmatic reticulum (ER).
• The term "sulfhydryl oxidase" was introduced more than 50 years ago.
Ero1 vs QSOX1 Two distinct flavin-linked sulfhydryl oxidase families: Ero1 and Quiescin-sulfhydryl oxidase QSOX1
protein disulfide isomerase (PDI): contains two CxxC redox-active disulfides (4 -SH equivalents upon reduction)
The present work
Widely-used model protein: pancreatic RNase, with 4
disulfides and 105 fully oxidized disulfide isomers.
A more complicated model: riboflavin binding protein (RfBP,
a protein with 9 disulfides and hence >34 million pairings for
the fully oxidized protein).
Quenching of riboflavin fluorescence upon binding to the
folded apoprotein allows continuous monitoring of oxidative
folding.
PROCEDURES• Subcloning, Expression, and Purification of Human (gi
48735337) and Chicken (gi 30923135) PDI
• Preparation of reduced and oxidized proteins (PDI, RNase, RfBP)
• Monitoring QSOX-mediated thiol oxidation using DTNB
• Refolding of Rnase followed by hydrolysis of cyclic CMP
• Refolding of RfBP followed by fluorescence
• Calculation of redox state of a and a' domains of PDI in
equilibrium with glutathione redox buffer
RESULTS
Human and avian PDI are poor substrates of avian QSOX1
Symbol QSOX/PDI
☐ Avian/human
✪ Avian/avian
⏏ Human/human
+× control
Oxidative refolding of pancreatic RNase with QSOX, PDI and redox buffers
SymbolReduced RNase
incubated with …
reduced PDI, QSOX
⏏ QSOX
✪ reduced PDI, GSH, GSSG
GSH, GSSG
Oxidative refolding of reduced riboflavin binding protein (RfBP)
Refolding of reduced RfBP in the absence of QSOX
SymbolReduced RfBP incubated with riboflavin in the presence of…
redox buffer of GSH/GSSG
⏏ redox buffer, reduced PDI
redox buffer, reduced PDI,
QSOX
QSOX, reduced PDI
Oxidative refolding of reduced RfBP using PDI alone
CONCLUSIONS
Whether oxidizing equivalents are
generated by QSOX, Ero1, or other cellular
oxidants, the universal additional
requirement for efficient oxidative folding is
reduced PDI.
Our PDIs are quite similar~!