The genes encoding PDI, abb′x, bb′xa′ are cloned into pQE30 (Qiagen), and the resulting proteins contain N-terminal (MRGSH₆GS) tags. The PDI gene was also cloned into pET24a (Novagen), and the resulting protein contained the C-terminal (His₆) tag. Genes encoding PDI, W111F/W390F PDI, W111F abb′x, and b′x are cloned into pET23b (Novagen), and the resulting proteins contained (MH₆M) as the N-terminal tags. The I272A and L343A mutants were constructed on the plasmids of their corresponding background proteins by overlap extension PCR (for pQE30) or site-directed mutagenesis (for pET23b), and the sequences of all the constructs were verified by DNA sequencing. Some of the above clones were kindly provided by Prof. L. W. Ruddock (University of Oulu). Proteins from pQE30 (18) or pET23b (16) were expressed and purified as described previously. Before further investigation, all preparations were assessed by native PAGE to confirm that they were monomeric (16). Protein concentrations were determined by the Bradford method with bovine serum albumin as a standard (21) or spectrophotometrically at 280 nm with the absorption coefficients 45.38 mm⁻¹cm⁻¹ for PDI and its I272A and L343A mutants, 34.76 mm⁻¹cm⁻¹ for W111F/390F PDI and its corresponding mutants, and 24.41 mm⁻¹cm⁻¹ for W111F abb′x and its mutants.

PDI proteins were digested by proteases in 50 mm Tris-HCl buffer (pH 7.6) containing 0 mm (for trypsin), 5 mm (for proteinase K), or 10 mm CaCl₂ (for chymotrypsin) and 10 mm dithiothreitol (for proteinase K and chymotrypsin) or 1 mm GSH (for trypsin). The reactions were terminated by phenylmethanesulfonyl fluoride and analyzed by reducing SDS-PAGE.

Protein samples were desalted into 100 mm ammonium acetate using a BioMax Ultrafree-0.5 PBCC centrifugal filter unit with a 5-kDa nominal cut-off. Six dilutions (10-fold) and concentration steps were performed. All data were acquired on a calibrated quadrupole-time of flight (Q-ToF I) instrument operated in positive ion mode with a precision of 3.5 ppm over the m/z range 500–3000. Each sample was manually infused using a nanoflow capillary after a 2-fold dilution in 30% acetonitrile, 1% formic acid.

Coomassie-stained gel pieces were processed and trypsin-digested using the manufacturer's recommended protocol on the MassPrep robotic protein handling system. The extracted peptides from each sample were analyzed by means of nanoliquid chromatography (LC)-electrospray ionization-MS/MS using a 45-min LC gradient. All MS and MS/MS data were corrected for mass drift using reference data collected from [Glu¹]-Fibrinopeptide B (human, F3261; Sigma) sampled each minute of data collection. The data were used to interrogate the UniProt data base release 15.5 appended with the two PDI sequences supplied using ProteinLynx Global Server Version 2.3. Data base search parameters used included chymotrypsin and proteinase K as the primary reagent and trypsin as the secondary reagent. Each sample was analyzed twice; the first injection used 4.9 μl of the digest solution, and the second was ∼25 μl to maximize the numbers of identified peptides.

Intrinsic fluorescence spectra of 5 μm proteins in 50 mm Tris-HCl buffer containing 150 mm NaCl (pH 7.6) were recorded at 310–400 nm at 25 °C with excitation at 290 nm.

ANS was added to 50 μm in 50 mm Tris-HCl buffer containing 150 mm NaCl (pH 7.6) in the absence or presence of 5 μm PDI proteins and incubated for 20 min at 25 °C in the dark before the ANS fluorescence emission spectra (400–600 nm) were determined with excitation at 370 nm. The concentration of ANS was determined using an extinction coefficient at 350 nm of 5000 m⁻¹cm⁻¹ (22).

Quenching experiments were performed by repeatedly adding 25 μl of 2 m potassium iodide (KI) to a 1-ml sample containing 5 μm purified protein in 20 mm phosphate buffer (pH 7.3) up to a final concentration of 0.33 m potassium iodide (8 additions). KI was also added to 1 ml of buffer as a blank. All fluorescence measurements were carried out in a cuvette with a magnetic stir bar to ensure thorough mixing after each addition, and determinations of intrinsic fluorescence intensity were corrected for dilution. Because the plot of F₀/F (where F₀ and F are, respectively, the fluorescence intensities at maximum emission wavelength of protein in the absence and presence of quencher) against [KI] was linear in every case, static quenching could be ignored, and the modified form of the Stern-Volmer equation (23, 24), F₀/F = (1 + K_sv[KI])e^V[KI], which includes the static quenching constant V, could be simplified as F₀/F = 1+ K_sv[KI]. The Stern-Volmer constant (K_sv) for dynamic quenching was determined as the slope of the above plot.

CD spectra were measured at 25 °C in a 0.1-cm path length thermostatically controlled cuvette using a Pistar-180 instrument (Applied Photophysics). The spectra were acquired at 190–260 nm using a 1-nm step size and 2-nm slit widths. For heat-induced protein denaturation, CD signals at 222 nm were collected from 25 to 85 °C with 2 °C intervals controlled by a thermoelectric temperature controller. The real temperature in the cuvette was detected by an inner probe. All CD data were averages of five scans.

Complete denaturation of rhodanese was carried out by incubation of 45 μm rhodanese in 6 m guanidine hydrochloride with 10 mm dithiothreitol for 1 h at room temperature. Refolding was initiated by 100-fold dilution of the denatured rhodanese into a 200 mm sodium phosphate buffer (pH 7.5) in the absence or presence of 2.25 μm PDI proteins at 20 °C. In another assay, 90 μm native rhodanese with 10 mm Na₂S₂SO₃ was 60-fold diluted into the same phosphate buffer without or with 24 μm PDI proteins equilibrated in a water bath of 40 °C. Aggregation was monitored by recording the light scattering at 350 nm in both assays, and the suppression of aggregation during the refolding or heating was used to measure the chaperone activity of PDI proteins (25).

Denatured and reduced RNase A, prepared as described (26), was reactivated at 8 μm in glutathione redox buffer (100 mm Tris acetate buffer containing 50 mm NaCl, 1 mm EDTA, 1 mm GSH, and 0.2 mm GSSG (pH 8.0)) in the presence of 3 μm PDI proteins at 25 °C, and RNase activity was determined by monitoring the absorbance increase at 296 nm due to hydrolysis of 4.5 mm cCMP. The slope of the initial linear increase in RNase activity after the lag time is taken as the measure of isomerase activity. The relative activity (%) was calculated as (A − A₀)/(A₁ − A₀) × 100%, where A is the activity of PDI mutants or abb′x mutants, A₁ is the activity of PDI or abb′x, and A₀ is the blank in the absence of protein.

Insulin of 130 μm was added to 0.1 m potassium phosphate buffer containing 2.5 mm EDTA, 0.1 mm dithiothreitol (pH 7.5) in the absence or presence of 0.5 μm PDI/mutants or 2.5 μm abb′x/mutants to initiate the reaction, and the absorbance at 650 nm that represents light scattering from reduced and precipitated insulin chains was immediately recorded at 25 °C. The activity was calculated by the maximal slope of the curve relative to the lag time (27).

We examined the intrinsic molecular flexibility in hPDI by limited proteolysis studies using proteinase K, chymotrypsin, and trypsin and working with full-length PDI and with species lacking specific domains. All constructs contained His₆ tags, which facilitated analysis of the location of cleavages by blotting with anti-His₆ antibodies.

As shown in Fig. 2A, digestion of full-length PDI (abb′xa′c) with proteinase K generated three new fragments, β, γ, and δ (lanes 6 and 9). The two smaller PDI fragments (γ, δ) could be the fragments abb′x and α (a digestion product of abb′x), respectively, in terms of their mobility in SDS-PAGE (compare lanes 6 to 4). No band corresponding to the size of bb′xa′ appeared in the digestion, implying that the a′ domain was much more sensitive than the a domain to be removed from PDI. Digestions of recombinant fragments abb′x and bb′xa′ were performed for comparison. The abb′x construct is quite resistant to proteinase K, giving rise over time to a slightly smaller product (α) (compare lanes 1, 4, and 7). When bb′xa′ was treated under the same conditions, no large product bands were detectable by Coomassie Blue with the exception of a faint band (ϵ) at around 25 kDa, although quantitative analysis of the bb′xa′ band intensity showed a decrease of 42% at 30 min (lane 8).

Because all of these PDI preparations contain a (MRGSH₆GS) tag at the N terminus, blotting with anti-His₆ monoclonal antibody was used to analyze the above profiles (Fig. 2B). All the bands detectable by Coomassie Blue in Fig. 2A were also visible in Fig. 2B, indicating that the N terminus is intact in every case and that proteinase K has removed varying amounts of sequence from the C terminus of these constructs. A further band (θ) was visible in the blot (lanes 5 and 8) as a digestion product of bb′xa′ slightly larger than ϵ but was not observed in the dye-stained gel. The data show clearly that exposure of PDI to proteinase K gives rise to a series of degradation products in which regions have been removed from the C terminus, but no large non-His₆ products are observed, indicating that there is little degradation of the N-terminal region. The relative molecular masses of the various constructs and proteinase K-derived fragments were estimated by mobility on SDS-PAGE, and all the products can be rationalized on the basis of cleavages within the C-terminal tail region (β) or within the x-linker (supplemental Fig. S1).

As an independent check, a PDI construct with a different N-terminal tag, MH₆M, was digested with proteinase K to generate an SDS-PAGE profile (supplemental Fig. S2A) in which a band of mobility comparable to band δ (Fig. 2A) was the most prominent product. The digest after 120 min was then analyzed by electrospray mass spectrometry, and the major product was found to have a mass of 38,035 Da, consistent with cleavages after two Met residues at residue −1 (i.e. at the junction between the N-terminal tag and the mature protein) and at residue Met-339 in the x-linker region (note that there was no Met at position −1 in the material analyzed in Fig. 2). This identification was confirmed by mass spectrometric analysis of tryptic digests of this product, which revealed peptides from the a, b, and b′ domains of PDI but none from the a′ domain (data not shown). It was further confirmed by the fact that proteinase K digestion of a full-length L343A mutant PDI (supplemental Fig. S2B) gave rise to a most prominent product of identical mass, indicating that the 38,035-Da product did not extend as far as residue 343.

To avoid conclusions based on the specificity of a single protease, we also carried out the digestion of hPDI by chymotrypsin. As shown in Fig. 2C, two major bands (2 and 3) and three weak bands (1, 4, and 5) appeared. The two major bands (2 and 3) around 45 kDa were both clearly recognized by anti-His₆ monoclonal antibody (Fig. 2D), and bands 1 and 4 were also recognized by anti-His₆ antibody after a longer time for exposure and development of the blot (data not shown), suggesting that all these large fragments (>35 kDa) still contain domain a at their N terminus and that the principal site(s) of cleavage by chymotrypsin is also in the C-terminal half of the molecule. Again as an independent check, the PDI construct with the MH₆M N-terminal tag was digested with chymotrypsin to generate an SDS-PAGE profile in which a band around 45 kDa was the most prominent product (supplemental Fig. S2C). The digest after 120 min was then analyzed by mass spectrometry, and the major product was found to have a mass of 43,873 Da corresponding exactly to that of the product extending from the N-terminal tag through to Trp-379, adjacent to the active site in the a′ domain. Identification of the cleavage site as occurring at this position within the a′ domain was confirmed by two observations. First, mass spectrometric analysis of tryptic digests of this major product contained many tryptic peptides lying N-terminal to this site but none from locations C-terminal to this site (data not shown). Second, analysis of a chymotrypsin digestion of a corresponding full-length PDI containing the L343A mutation (supplemental Fig. S2D) gave rise to a major product of mass 43,833; the difference in mass of 40 between this product and that from wild-type PDI confirms that the site of cleavage is C-terminal to the position of this mutation.

In addition, we carried out digestion of hPDI by trypsin. As shown in Fig. 2E, with 500 μg/ml trypsin hPDI was hydrolyzed into 5 major components. Three bands above 34 kDa (a, b, and c) were recognized by anti-His₆ monoclonal antibody (Fig. 2F) and must be fragments with domain a intact but a′ or part of a′ trimmed off. The two smaller fragments (d and e) were not recognized by anti-His₆ antibody; band e (∼15 kDa) may be a single domain, and band d (∼24 kDa) may represent bb′. This result is consistent with that from using the other proteases and confirms that interdomain mobility in hPDI is more apparent in the b′xa′ region than in the ab region.

To check the possibility that the N-terminal (MRGSH₆GS) tag protected the a domain from digestion, we removed this tag and made a new construct of PDI with a C-terminal (His₆) tag. The major product of this protein digested by chymotrypsin has similar mobility to that of PDI with the MRGSH₆GS tag at the N terminus (supplemental Fig. S3), and this product cannot be recognized by anti-His₆ tag antibody (data not shown), indicating that the principal digestion sites are unaffected and not dependent on the location of the tag.

Having established from protease studies that there is flexibility in the b′xa′ region of full-length PDI, we aimed to define the nature of the underlying conformational changes. Previous work on b′x and bb′x constructs has exploited the fluorescence and NMR properties of the unique tryptophan residue (Trp-347) in the x-linker as a conformation-sensitive probe and has established that the x-linker can adopt alternative conformations with respect to the b′ domain, either bound into the ligand binding pocket on b′ to give a capped conformation or displaced from this pocket, giving an uncapped conformation (15). We wished to determine if such alternative conformations occur in full-length PDI, building on the observation that the NMR properties of the indole NH of Trp-347 in full-length PDI indicate that it is mobile between alternative conformations (15).

In full-length PDI there are five tryptophan residues, Trp-35 and Trp-111 in the a domain, Trp-379 and Trp-390 in the a′ domain, and Trp-347 in the x region (see Fig. 1). The fluorescence of Trp-35 has been shown to be heavily quenched by the adjacent active site of the a domain in both dithiol and disulfide states (28), and we hypothesized that the fluorescence of Trp-379 in the a′ domain, which is situated just before the active site in an analogous position to Trp-35, would also be quenched. Hence, we constructed a double-mutant full-length PDI (W111F/W390F) in the hope that its fluorescence would be dominated by Trp-347 that could then act as an unambiguous probe of the conformation of x-linker in full-length PDI.

As shown in Fig. 3A, the intensity of the emission spectrum of W111F/W390F PDI is similar to that of b′x and is much lower than that of wild-type PDI. This is consistent with the suggestion that the fluorescence of W111F/W390F PDI is mainly contributed by Trp-347 in the x region. In isolated b′x, the I272A and L343A mutants have been shown to shift the conformation of the x-linker to the capped and uncapped conformation, respectively (15). Consequently, we constructed the I272A and L343A mutants of full-length PDI in the W111F/W390F mutant background and used their intrinsic fluorescence as models to investigate conformational change and the existence of alternative conformations (capped and uncapped) in full-length PDI.

The intrinsic fluorescence spectra of W111F/W390F PDI and its I272A and L343A mutants were measured with excitation at 290 nm (Fig. 3B). The maximal emission wavelength of W111F/W390F PDI is 344 nm. The I272A mutant shows a blue shift with a emission maximum at 341 nm, and the L343A mutant shows a red shift with the emission maximum at 347 nm, suggesting that the shifts of conformation to a capped state by I272A and uncapped state by L343A observed in b′x also exist in full-length PDI. Because the maximum of fluorescence emission is difficult to determine precisely and does not necessarily define the properties of the full emission spectrum, we also used the full emission data from 310 to 400 nm to calculate the barycentric mean emission wavelength, the “center of gravity” of the spectrum, as an objective measure of spectral shift on mutants. This analysis confirmed that effects of the mutations in full-length W111F/W390F PDI were in the same directions as observed for b′x but smaller in magnitude (Table 1).

Fluorescence quenching experiments are an effective method for measuring the exposure to solvent of fluorophores within proteins, and hence, we used quenching of fluorescence by I⁻ as a probe of tryptophan exposure in wild-type b′x in full-length W111F/W390F PDI and in their I272A and L343A mutants. Over the quencher range used, Stern-Volmer plots were linear (e.g. supplemental Fig. S4) and were analyzed to give simple dynamic quenching constants (K_sv). In the b′x background, the K_sv values of the wild-type and the I272A mutant are similar, 0.92 and 1.03 m⁻¹ (Table 1), but a considerably larger value 2.14 m⁻¹ was observed for the L343A mutant, consistent with Trp-347 being more exposed in the uncapped L343A mutant, as its fluorescence is more easily quenched. Similar results are also observed in the full-length protein. In W111F/W390F PDI background, the K_sv for the L343A mutant is again significantly higher than that for the background and for I272A mutant. These findings demonstrate that the L343A mutation in full-length PDI causes more exposure of Trp-347 compared with wild-type or I272A mutant, suggesting that this mutation promotes the uncapped conformation in full-length PDI.

We also used the less specific methods of ANS fluorescence and limited proteolysis to probe the conformational changes generated in full-length PDI by the I272A and L343A mutations. As shown in Fig. 4A and Table 2, wild-type PDI and L343A PDI show similar ANS fluorescence intensity, but I272A PDI shows lower ANS fluorescence intensity, indicating less hydrophobic area exposed, consistent with adopting the capped conformation in which the hydrophobic ligand binding site is obstructed. In the limited proteolysis assay, the full-length L343A PDI band almost disappeared after 21 min of digestion by proteinase K (Fig. 5A), whereas about half of I272A PDI and wild-type PDI is intact, indicating that L343A PDI is more sensitive to proteinase K than I272A PDI and wild-type PDI, consistent with a greater tendency to adopt an uncapped conformation. I272A PDI and PDI show similar protease sensitivity over this time scale; a band just below the full-length protein band becomes prominent (cf. band β in Fig. 2, A and B), possibly indicating removal of the C-terminal tail region. Digestion profiles of mutants of full-length PDI by chymotrypsin (Fig. 5B) gave similar results with the L343A mutant much more sensitive to this protease.

We noticed that the shifts in fluorescence produced by I272A and L343A mutations are quantitatively smaller in full-length PDI (W111F/W390F) than in isolated b′x (Table 1 and Ref. 15), suggesting that the conformational change is moderated by the effects of the adjacent domains. We assumed that the presence of the a′ domain may affect the conformation of the x-linker. To address this question, we constructed the W111F abb′x truncation and its I272A and L343A mutants for fluorescence study of Trp-347. The maximal emission wavelength of W111F abb′x was 339 nm (Fig. 3C), which is a blue shift compared with that of W111F/W390F PDI (344 nm), indicating a more hydrophobic environment for Trp-347 in the truncated molecule with a′ domain removed. This conclusion is confirmed by the analysis of the barycentric mean emission wavelengths (Table 1). In this background the I272A mutation produces W111F/I272A abb′x, which shows emission maximum at 334 nm, a blue shift compared with 339 nm for W111F abb′x, whereas W111F/L343A abb′x shows a red shift with emission maximum at 342 nm; again, these results are confirmed by the analysis of barycentric mean emission wavelength (Table 1).

No fluorescence quenching by iodide was detected for W111F abb′x over the quencher concentration range used, indicating that the Trp-347 residue is buried and inaccessible in this truncated protein. However, the quenching of the Trp-347 in full-length PDI was detected to be 0.84 m⁻¹. The above data indicate that the Trp-347 is more exposed to aqueous medium in the full-length protein, very likely due to the existence of the a′ domain, which makes the x-linker favor the uncapped conformer. The quenching experiments showed significant quenching of Trp-347 fluorescence in the W111F/L343A mutant of abb′x but not in W111F abb′x and the I272A mutant, also indicating that in abb′x the L343A mutation can induce formation of the uncapped state, with Trp-347 more exposed.

We also examined the hydrophobic exposure of abb′x and its I272A and L343A mutants by ANS fluorescence. As shown in Fig. 4B, I272A abb′x displays a much lower ANS fluorescence intensity than that of abb′x, indicating a capped conformation. L343A abb′x shows a similar ANS fluorescence intensity to abb′x. Comparison of the ANS enhancements for I272A abb′x and for I272A PDI (Table 2) suggests that the former is almost entirely in the capped conformation, which is prevented by the a′ domain in the full-length molecule.

From Fig. 2, A and B, it is known that abb′x is digested at its C terminus by proteinase K to give a slightly smaller product (α). In Fig. 5C, the effects of I272A and L343A mutations on proteinase K sensitivity of abb′x were explored. The upper band corresponding to abb′x disappeared by 9 min in abb′x but persisted in I272A abb′x, whereas L343A abb′x completely disappeared by 6 min of digestion. These results are consistent with the expected mutation-induced shifts between capped and uncapped conformations. But the fact that the difference observed in abb′x background is more marked than in full-length PDI background (Fig. 5, A and B) again demonstrates that the truncated protein shows clearer differentiation of capped and uncapped states than does full-length PDI.

To ensure our conclusions are not due to possible gross structural effects of PDI mutations, we measured the far-UV CD spectra of all the mutants. The mutants in either PDI (supplemental Fig. S5A) or abb′x (supplemental Fig. S5B) background showed very similar far-UV CD spectra, indicating that none of the mutations affect the secondary structures of PDI or abb′x. Thus, the Trp-to-Phe mutant proteins are good models for the background proteins while providing more site-specific intrinsic fluorescence probes of conformational change and environment of the x-linker. In addition, we tested the thermal stability of I272A and L343A mutants in both PDI (Fig. 6A) and abb′x backgrounds (Fig. 6B). The heat-induced unfolding curves of I272A and L343A mutants are also very similar to those of the background proteins, suggesting that the I272A and L343A mutations did not destabilize the structure of the background proteins. Hence, the differences we observed with the I272A and L343A mutations in the fluorescence and limited digestion assays arise from their effects on the balance between the capped and uncapped conformations. Furthermore, we found that full-length PDI (Fig. 6A) gradually loses a fraction of its secondary structure as the temperature rises from 25 to 50 °C, whereas the secondary structure of abb′x (Fig. 6B) is quite stable in the same temperature range and shows a single cooperative transition at around 60 °C. This indicates that the a′ domain is relatively unstable compared with abb′x, which is consistent with our finding that the a′ domain is more susceptible to digestion than the a domain.

We next investigated the functional significance of these alternative conformations. As shown in Fig. 7A, I272A PDI showed significantly lower potency than PDI in suppressing aggregation of denatured rhodanese upon dilution, suggesting that less hydrophobic area was exposed and available for binding the rhodanese refolding intermediate in the I272A PDI molecule with the capped conformation. L343A PDI showed the same chaperone activity as that of PDI. abb′x and its mutants did not show measurable chaperone activity under these conditions (data not shown) but did show significant activity in an alternative assay, the ability to suppress heat-induced aggregation of rhodanese (Fig. 7B) with I272A abb′x showing lowest ability.

We also tested the protein disulfide isomerase (Fig. 8) and protein disulfide reductase (Fig. 9) activities of PDI and its mutants. The I272A and L343A mutants of full-length PDI are quite similar in both activities to wild-type PDI. However, marked differences were observed in the abb′x background. I272A abb′x showed significantly lower isomerase (43%) and reductase (21%) activity, while L343A abb′x showed higher isomerase (116%) and reductase (226%) activity.