Expression and purification of recombinant rat protein disulfide isomerase from Escherichia coli

Self Cite

130

Protein-disulfide isomerase (PDI) catalyzes the formation and isomerization of disulfides during oxidative protein folding. This process can be error-prone in its early stages, and any incorrect disulfides that form must be rearranged to their native configuration. When the second cysteine (CGHC) in the PDI active site is mutated to Ser, the isomerase activity drops by 7-8-fold, and a covalent intermediate with the substrate accumulates. This led to the proposal that the second active site cysteine provides an escape mechanism, preventing PDI from becoming trapped with substrates that isomerize slowly (Walker, K. W., and Gilbert, H. F. (1997) J. Biol. Chem. 272, 8845-8848). Escape also reduces the substrate, and if it is invoked frequently, disulfide isomerization will involve cycles of reduction and reoxidation in preference to intramolecular isomerization of the PDI-bound substrate. Using a gel-shift assay that adds a polyethylene glycol-conjugated maleimide of 5 kDa for each sulfhydryl group, we find that PDI reduction and oxidation are kinetically competent and essential for isomerization. Oxidants inhibit isomerization and oxidize PDI when a redox buffer is not present to maintain the PDI redox state. Reductants also inhibit isomerization as they deplete oxidized PDI. These rapid cycles of PDI oxidation and reduction suggest that PDI catalyzes isomerization by trial and error, reducing disulfides and oxidizing them in a different configuration. Disulfide reduction-reoxidation may set up critical folding intermediates for intramolecular isomerization, or it may serve as the only isomerization mechanism. In the absence of a redox buffer, these steady-state reductionoxidation cycles can balance the redox state of PDI and support effective catalysis of disulfide isomerization.The folding of proteins destined for the secretory pathway occurs in the endoplasmic reticulum where a quality control system ensures that secreted proteins are correctly folded (1), including the correct formation of disulfide bonds. Disulfides that form early in the folding process are often incorrect; cysteines can be mispaired (2), or disulfides can be formed in the wrong temporal order, making it difficult to oxidize buried cysteines (3). To rectify these errors, the incorrect disulfides must be broken and new ones formed in a different configuration.The endoplasmic reticulum contains folding assistants that help proteins achieve their correct disulfide arrangement. Protein-disulfide isomerase (PDI) 1 is a 55-kDa protein of the endoplasmic reticulum which catalyzes disulfide formation (oxidase activity) as well as the rearrangement of incorrect disulfide pairings (isomerase activity) (4), accelerating both processes without drastically altering the refolding pathway (1). PDI catalyzes the chemical changes but does not appear to guide the process or unfold misfolded substrates actively (5, 6). PDI has two active sites, one near the amino terminus and the other near the carboxyl terminus (7). Each active site contains two cysteines in...

Section: Methodsmentioning

confidence: 99%

Reduction-Reoxidation Cycles Contribute to Catalysis of disulfide Isomerization by Protein-disulfide Isomerase

Schwaller¹,

Wilkinson²,

Gilbert

2003

Self Cite

130

“…Materials-Recombinant rat PDI was expressed and purified as described previously (34). The concentration of PDI monomers (55 kDa) was determined by absorbance at 280 nm (E 0.1% ϭ 0.94 cm Ϫ1 ml/mg) (35).…”

Section: Methodsmentioning

confidence: 99%

Hormone Binding by Protein Disulfide Isomerase, a High Capacity Hormone Reservoir of the Endoplasmic Reticulum

Primm

Gilbert

2001

Self Cite

Protein disulfide isomerase (PDI) is a folding assistant of the eukaryotic endoplasmic reticulum, but it also binds the hormones, estradiol, and 3,3,5-triiodo-L-thyronine (T 3 ). Hormone binding could be at discrete hormone binding sites, or it could be a nonphysiological consequence of binding site(s) that are involved in the interaction PDI with its peptide and protein substrates. Equilibrium dialysis, fluorescent hydrophobic probe binding (4,4-dianilino-1,1-binaphthyl-5,5-disulfonic acid (bis-ANS)), competition binding, and enzyme activity assays reveal that the hormone binding sites are distinct from the peptide/protein binding sites. PDI has one estradiol binding site with modest affinity (2.1 ؎ 0.5 M). There are two binding sites with comparable affinity for T 3 (4.3 ؎ 1.4 M). One of these overlaps the estradiol site, whereas the other binds the hydrophobic probe, bis-ANS. Neither estradiol nor T 3 inhibit the catalytic or chaperone activity of PDI. Although the affinity of PDI for the hormones estradiol and T 3 is modest, the high local concentration of PDI in the endoplasmic reticulum (>200 M) would drive hormone binding and result in the association of a substantial fraction (>90%) of the hormones in the cell with PDI. High capacity, low affinity hormone sites may function to buffer hormone concentration in the cell and allow tight, specific binding to the true receptor while preserving a reasonable number of hormone molecules in the very small volume of the cellular environment.

“…Overnight cultures from a single colony were grown on LB to an A 650 of 0.8 -1.0 and induced with 0.4 mM isopropyl-1-thio-␤-D-galactopyranoside (final concentration). After overnight growth, cells were harvested by centrifugation, disrupted as described previously (26), dialyzed, and applied to a DEAE-Sephacel column (2.5 ϫ 20 cm). weePDI was separated from wild-type PDI using a 2-liter linear gradient (total volume) of 0 -0.5 M NaCl in 0.025 M sodium phosphate, pH 6.3. weePDI and the various weePDI mutants eluted after the corresponding full-length PDI.…”

Section: Methodsmentioning

confidence: 99%

“…Expression and Purification-The expression of weePDI accompanies the expression of full-length PDI when expression is induced from expression plasmid (pET-PDI.2) (26). E. coli strain BL21(DE3) (Novagen, Madison, WI) was transformed with a plasmid encoding the wildtype PDI or various cysteine mutants (18).…”

Section: Methodsmentioning

confidence: 99%

A 21-kDa C-terminal Fragment of Protein-disulfide Isomerase has Isomerase, Chaperone, and Anti-chaperone Activities

Puig

Primm

Rajendran

et al. 1997

Self Cite

A catalyst of disulfide formation and isomerization during protein folding, protein-disulfide isomerase (PDI) has two catalytic sites housed in two domains homologous to thioredoxin, one near the N terminus and the other near the C terminus. The thioredoxin domains, by themselves, can catalyze disulfide formation, but they are unable to catalyze disulfide isomerizations (Darby, N. J. and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). A 21-kDa, C-terminal fragment of PDI (amino acids 308 -491), termed weePDI, comprises the C-terminal third of the molecule. The k cat for ribonuclease oxidative folding by weePDI is 0.26 ؎ 0.02 min ؊1 , 3-fold lower than the wild-type enzyme but indistinguishable from the activity of a full-length mutant of PDI in which both active site cysteines of the N-terminal thioredoxin domain have been mutated to serine. Eliminating the ability of weePDI to escape easily from covalent complexes with substrate by mutating the active site cysteine nearer the C terminus to serine has a large effect on the isomerase activity of weePDI compared with its effect on the full-length enzyme. weePDI also displays chaperone and anti-chaperone activity characteristic of the full-length molecule. As isolated, weePDI is a disulfide-linked dimer in which the single cysteine (Cys-326) outside active site cross-links two weePDI monomers. The presence of the intermolecular disulfide decreases the activity by more than 2-fold. The results imply that the functions of the core thioredoxin domains of PDI and other members of the thioredoxin superfamily might be modified quite easily by the addition of relatively small accessory domains. Protein-disulfide isomerase (PDI)1 is an abundant, 55-kDa resident of the endoplasmic reticulum (ER) (1-3). The enzyme catalyzes a variety of thiol/disulfide exchange reactions, including the formation and rearrangement of disulfide bonds during oxidative protein folding. PDI possesses two domains that are homologous to the redox active protein, thioredoxin (4). One is located near the N terminus, and another is found near the C terminus. Each of the domains is characterized by a redoxactive dithiol/disulfide site with the general sequence CGHC. Thioredoxin domains, including active-site CXXC sequences, have been found in a number of redox-active proteins, suggesting that these proteins constitute a thioredoxin superfamily with versatile thiol/disulfide exchange activities including oxidation, reduction, and isomerization (5).Individual members of the thioredoxin superfamily have up to three thioredoxin domains linked together. Erp72 has three thioredoxin domains (6), whereas Erp61 has two (7). Other members of the thioredoxin superfamily such as thioredoxin itself, glutaredoxin (8), or the Escherichia coli periplasmic protein, DsbA (9), have only one thioredoxin homology domain. All of these single domain proteins are poor catalysts of disulfide rearrangements, suggesting that domains other than the thioredoxin domains contribute significantly to substrate interactions that...