1. 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

2. 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 + O 2 → RS-SR + H 2 O 2 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.

3. 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)

5. 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.

6. 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

7. RESULTS Human and avian PDI are poor substrates of avian QSOX1 SymbolQSOX/PDI ☐ Avian/human ✪ Avian/avian Human/human +×control

8. Oxidative refolding of pancreatic RNase with QSOX, PDI and redox buffers Symbol Reduced RNase incubated with … reduced PDI, QSOX QSOX ✪ reduced PDI, GSH, GSSG  GSH, GSSG

9. Oxidative refolding of reduced riboflavin binding protein (RfBP)

11. Refolding of reduced RfBP in the absence of QSOX Symbol Reduced 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

12. Oxidative refolding of reduced RfBP using PDI alone

13. CONCLUSIONS Whether oxidizing equivalents are generated by QSOX, Ero1, or other cellular oxidants, the universal additional requirement for efficient oxidative folding is reduced PDI.

14. Our PDIs are quite similar~!