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~!