Pumtiwitt C. Rancy and Colin Thorpe *
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Transcript of Pumtiwitt C. Rancy and Colin Thorpe *
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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
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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.
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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)
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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.
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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
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RESULTS
Human and avian PDI are poor substrates of avian QSOX1
Symbol QSOX/PDI
☐ Avian/human
✪ Avian/avian
⏏ Human/human
+× control
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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
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Oxidative refolding of reduced riboflavin binding protein (RfBP)
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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
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Oxidative refolding of reduced RfBP using PDI alone
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CONCLUSIONS
Whether oxidizing equivalents are
generated by QSOX, Ero1, or other cellular
oxidants, the universal additional
requirement for efficient oxidative folding is
reduced PDI.
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Our PDIs are quite similar~!