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. 2001 Nov;10(11):2346–2353. doi: 10.1110/ps.23301

Chaperone-like activity of peptidyl-prolyl cis-trans isomerase during creatine kinase refolding

Wen-Bin Ou 1, Wei Luo 1, Yong-Doo Park 1,2, Hai-Meng Zhou 1
PMCID: PMC2374073  PMID: 11604540

Abstract

Porcine kidney 18 kD peptidyl-prolyl cis-trans isomerase (PPIase) belongs to the cyclophilin family that is inhibited by the immunosuppressive drug cyclosporin A. The chaperone activity of PPIase was studied using inactive, active, and alkylated PPIase during rabbit muscle creatine kinase (CK) refolding. The results showed that low concentration inactive or active PPIase was able to improve the refolding yields, while high concentration PPIase decreased the CK reactivation yields. Aggregation was inhibited by inactive or active PPIase, and completely suppressed at 32 or 80 times the CK concentration (2.7 μM). However, alkylated PPIase was not able to prevent CK aggregation. In addition, the ability of inactive PPIase to affect CK reactivation and prevent CK aggregation was weaker than that of active PPIase. These results indicate that PPIase interacted with the early folding intermediates of CK, thus preventing their aggregation in a concentration-dependent manner. PPIase exhibited chaperone-like activity during CK refolding. The results also suggest that the isomerase activity of PPIase was independent of the chaperone activity, and that the proper molar ratio was important for the chaperone activity of PPIase. The cysteine residues of PPIase may be a peptide binding site, and may be an essential group for the chaperone function.

Keywords: Peptidyl-prolyl cis-trans isomerase, creatine kinase, refolding, chaperone-like

Protein folding is often assisted by two kinds of factors in vivo: foldases, such as protein disulfide isomerase (PDI EC 5.3.4.1), and peptidyl-prolyl cis-trans isomerase (PPIase EC 5.2.1.8) and molecular chaperones (Freskgard et al. 1992). PDI accelerates and improves protein folding by reshuffling incorrect disulfide bridges and by its chaperone function. PPIase catalyzes the cis-trans isomerization of Xaa-Pro peptide bonds (where Xaa is the preceding amino acid) in oligopeptides and accelerates the slow rate-limiting steps in the refolding of several proteins in vitro (Fischer et al. 1984; Lang et al. 1987; Lin et al. 1988; Davis et al. 1989; Kiefhaber et al. 1990; Freskgard et al. 1992; Tan et al. 1997; Yang et al. 1997).

Porcine kidney PPIase was found to be identical with cyclophilin (CyP), which has the same N-terminus, binds the immunosuppressive drug cyclosporin A (CsA), and is inhibited by CsA (Fischer et al. 1989). PPIases are divided into three structurally unrelated families by their ability to specially bind immunosuppressive drugs: the CyP family, the FKBP (FK506 binding protein) family, and the parvulin family (Schmid 1998). PPIase is a ubiquitous, highly conserved, abundant 18-kD cytosolic protein (Hohman and Hultsch 1990). It has been found in all organisms and in different cell compartments such as the cytosol, the mitochondria, the endoplasmic reticulum and in the periplasm of Escherichia coli (Danielson et al. 1988; Davis et al. 1989; Haendler et al. 1989; Kawamukai et al. 1989; Lightowlers et al. 1989; Schneuwly et al. 1989; Shieh et al. 1989; Dietmeier and Tropschug 1990; Liu and Walsh 1990; Dartigalongue and Raina 1998). The various members of CyP family share a high degree of sequence similarity (Schonbrunner et al. 1991). Sequence comparison among CyPs revealed the presence of a conserved central core domain including a CsA binding site and a PPIase activity site, flanked in some cases by hydrophobic N- and C-terminal extensions. The N-terminal extension may serve as a signal sequence, and may be involved in the targeting of different PPIases to distinct subcellular compartments (Colley et al. 1991). The C-terminal extension is responsible for membrane anchoring of various PPIases (Arber et al. 1992). The X-ray crystal structure of human recombinant CyP is an eight-stranded antiparallel β-barrel structure (Kallen et al. 1991).

Studies have also shown that CyP exhibits a chaperone-like function in vitro. Using porcine kidney 18 kD PPIase, Freskgard et al. (1992) found that PPIase acted not only as a isomerase but also as a chaperone in the folding of carbonic anhydrase II (HCA II). Kern et al. (1994) repeated the above test with the refolding times lengthened from 1 to 4 h to show that PPIase accelerated the HCA II refolding speed, but was unable to protect the folding intermediates from aggregation. The N-terminal CyP-homologous domain of a 150-kD tumor recognition protein participates in the carbonic anhydrase folding process as a chaperone (Rinfret et al. 1994). In addition, two components of the mature Hsp90 complex, FKBP52 and P23, suppressed the aggregation of thermal unfolding citrate synthase in a concentration-dependent manner (Bose et al. 1996). The steroid aporeceptor complex contains the molecular chaperones Hsp90 and Hsp70, p48, CyP40, and the associated proteins p23 and p60, and in vitro β-galactosidase folding assays have shown that the CyP40 and p23 functions were similar to that of the molecular chaperone Hsp90 or Hsp70 (Freeman et al. 1996). Furthermore, MTFK (FK506 binding protein) suppressed the aggregation of folding intermediates and elevated the final yield of rhodanese refolding. The C-terminus played a more important role for the chaperon activity of MTFK (Furutani et al. 2000). Furthermore, FkpA markedly increased the yield of the single-chain Fv fragment in vitro. The FkpA folding-assisting function was independent of its isomerase activity (Ramm and Pluckthun 2000). Recently, hFKBP12, hCyP18, and bovine serum albumin (BSA) were reported to increase the yield of the antibody Fab fragment during refolding in vitro (Lilie et al. 1993). However, this effect was thought to be the result of nonspecific protein–protein interaction because CsA did not inhibit it (Furutani et al. 2000).

Understanding of the molecular mechanism of protein folding from a disordered polypeptide chain to the specific native state, deciphering of the second half of the genetic code, remains one of the major challenges in biochemistry (Kiefhaber et al. 1990). Creatine kinase (ATP: creatine N-phosphotransferase, CK, EC 2.7.2.3) is a key enzyme of cellular energy metabolism, which catalyzes the reversible phosphoryl transfer from phosphocreatine to ADP (Watt 1973; Jacobus 1985). Extensive investigations have been carried out to understand the folding mechanism of CK (Bickerstaff et al. 1980; Yao et al. 1984; Wang et al. 1995; Michael et al. 1998). Yang et al. (1997) studied the effect of pig kidney 18 kD PPIase on the refolding and reactivation of urea-denatured CK to show that PPIase accelerated the slow-phase reaction of the CK refolding. However, disagreement still exists over whether pig kidney PPIase exhibits chaperone activity (Freskgard et al. 1992; Kern et al. 1994). Therefore, this study used rabbit muscle CK as a target protein to investigate the chaperone activity of PPIase. The results showed that PPIase was able to markedly improve the reactivation yields and inhibited aggregation of CK. However, alkylated PPIase was unable to prevent CK aggregation. These results indicate that PPIase displayed chaperone-like activity during CK refolding.

Results

Activity assay of PPIase after being stored 2 years at −20°C

PPIase activity was assayed at 10°C in a chymotrypsin-coupled assay in a 50 mM Tris-HCl buffer (pH 7.5). Two different concentrations of PPIase were used for the activity assay: 16 and 100 μM. The enzymatic activity was measured using absorption at 390 nm. As shown in Figure 1 ▶, increasing concentrations of PPIase did not show any isomerase activity relative to the negative control with no PPIase and the positive control of 5 μM active PPIase.

Fig. 1.

Fig. 1.

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Activity assay of PPIase after being stored at −20°C for about 2 years. Enzymatic activity of PPIase after being stored at −20°C for about 2 years assayed at 10°C in a chymotrypsin-coupled assay in a 50-mM Tris-HCl buffer (pH 7.5). Two different concentrations of PPIase were used for the PPIase assay: 16 and 100 μM. The total volume of the reactive system was 400 μL. Curves 1, 3, and 4 represent 0 μM, 16 μM and 100 μM of PPIase. Curve 2 is the positive control of 5 μM active PPIase.

Intrinsic fluorescence emission spectra of inactive, active, and alkylated PPIase

The intrinsic fluorescence spectra of inactive and alkylated PPIase were measured over a wavelength range of 300–400 nm. The same concentration of active PPIase was used as the control. As shown in Figure 2 ▶, the peak positions of all samples were at 337 nm, but their fluorescence intensities differed. The intensity of alkylated PPIase was the lowest among the measured samples. The structure of inactive PPIase was similar to that of active PPIase.

Fig. 2.

Fig. 2.

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Intrinsic fluorescence emission spectra of inactive and alkylated PPIase. Inactive and alkylated PPIase concentrations were 20 μM. Active PPIase at the same concentration was used as the control. An excitation wavelength of 280 nm was used for measuring the fluorescence intensity. The reactions all occurred at 25°C. Curves 1, 2, and 3 represent active PPIase, inactive PPIase, and alkylated PPIase.

SEC of inactive and active PPIase

The SEC of inactive PPIase was similar to that of active PPIase (Fig. 3 ▶). Their elution volumes were all 18.5 mL. SEC data showed that the polypeptide chain was not degraded.

Fig. 3.

Fig. 3.

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SEC of inactive PPIase. SEC of inactive PPIase was measured with a superdex HR200. Active PPIase was used as the control. The SEC was recorded with an AKTA purifier. Curves 1 and 2 are active PPIase and inactive PPIase.

Reactivation of CK in the presence of PPIase

Denatured CK (150 μM) was diluted 136-fold or 75-fold with refolding buffer (30 mM Tris•HCl/1 mM EDTA pH 8.0) containing inactive PPIase or active PPIase. The CK activity was measured over time. The data showed that the self-reactivation enzymatic activity recovered rapidly to approximately 60–70% of the native CK activity. However, the CK reactivity suddenly increased about 20% upon the addition of low concentration inactive or active PPIase into the renaturation system compared to the self-renatured CK with no PPIase. The enzymatic activity gradually decreased with increased PPIase concentrations (Fig. 4 ▶). The enzyme activities of the terminal states are shown in Figure 5 ▶. The results showed that the active PPIase had a greater effect on CK reactivation than the inactive PPIase. The refolding yield of CK was maximized at a PPIase and CK molar ratio of 4:1.

Fig. 4.

Fig. 4.

Fig. 4.

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Effect of inactive or active PPIase on the reactivation time of GdmCl-denatured CK. The CK was denatured in 3 M GdmCl for 1 h at 25°C. The CK reactivation was initiated by diluting unfolded enzyme (150 μM) 136-fold or 75-fold into the standard buffer in the absence (filled circles) and presence of various amounts of inactive PPIase (A): 1.1 μM (X), 2.2 μM (filled diamonds), 4.4 μM (filled squares), 8.8 μM (open triangles), 17.6 μM (filled triangles), and 55 μM (+); and active PPIase (B): 2 μM (filled up triangles), 8 μM (filled diamonds), 16 μM (filled down triangles), and 32 μM (X). Enzymatic activity was measured at the indicated times. The native enzyme activity at the same concentration was taken as 100%.

Fig. 5.

Fig. 5.

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Comparison of the influence of inactive and active PPIase on the CK reactivation process. The data was from Figure 4 ▶. Experimental points show the remaining activity. Curve (filled squares) is the CK reactivity assisted by inactive PPIase, and curve (filled circles) is the CK activity assisted by active PPIase. The negative control points (filled triangles) are the activity of refolded CK in the presence of the same concentration of BSA.

Aggregation of CK in the presence of PPIase

In general, CK aggregation occurs in a concentration- and temperature-dependent manner (Li et al. 2001). When denatured enzyme (150 μM) was diluted into the refolding buffer used in the previous section, it aggregated immediately. However, aggregation of denatured CK added to the same buffer with different amounts of inactive or active PPIase as indicated in the figure legends was inhibited and almost suppressed at 80-fold or 32-fold concentrations of CK (2.7 μM) (Fig. 6 ▶). The maximum turbidity during CK aggregation in the absence and presence of inactive and active PPIase is shown in Figure 7 ▶. The results showed that the active PPIase more effectively prevented aggregation than the inactive PPIase at the same molar ratio. The alkylated PPIase was unable to prevent CK aggregation (Fig. 8 ▶).

Fig. 6.

Fig. 6.

Fig. 6.

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Suppression of CK aggregation by inactive or active PPIase during refolding. GdmCl-denatured CK (3 M) (150 μM) was diluted 55-fold with refolding buffer (30 mM Tris-HCl/1 mM EDTA [pH 8.0]) containing inactive PPIase (A) or active PPIase (B). The reactions all occurred at 37°C. Aggregation was monitored by measuring the turbidity at 400 nm. The refolding buffer for Figure 4A ▶ contained 1. 0 μM, 2. 5.4 μM, 3. 17.6 μM, 4. 43.2 μM, 5. 86.4 μM, 6. 135 μM, or 7. 216 μM inactive PPIase. The refolding buffer for Figure 4B ▶ contained 1. 0 μM, 2. 2.7 μM, 3. 5.4 μM, 4. 10.8 μM, 5. 21.6 μM, 6. 43.2 μM, or 7. 86.4 μM active PPIase.

Fig. 7.

Fig. 7.

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Comparison of the suppression of CK aggregation by inactive or active PPIase during refolding. Curve (filled squares) is the maximal turbidity of refolding CK assisted by inactive PPIase, and curve (filled circles) is the maximal turbidity of refolding CK assisted by active PPIase. The negative control points (filled triangles) are the turbidity of refolded CK in the presence of the same concentration of BSA.

Fig. 8.

Fig. 8.

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Influence of alkylated PPIase on CK aggregation. Denatured CK (150 μM) was diluted 55-fold with a refolding buffer (30 mM Tris-HCl/1 mM EDTA pH 8.0) containing various amounts of alkylated PPIase. Aggregation was monitored by measuring the turbidity at 400 nm. The reactions all occurred at 37°C. The experimental points (filled circles) show the turbidity of refolding CK assisted by alkylated PPIase. The control points (filled diamonds) show the turbidity of refolding CK assisted by active PPIase.

Discussion

A chaperone is able to transiently bind and thus stabilize an unstable conformation of another protein, thereby facilitating correct folding by preventing misfolding and aggregation (Rassow et al. 1997). Although a variety of mechanisms have been proposed for the chaperoning mechanism, some details in the folding process remain for further study.

In recent years, further examples of the chaperone function have been discovered such as CyP-40, FKBP52, and the trigger factor of E. coli (Bose et al. 1996; Freeman et al. 1996; Hesterkamp and Bukau 1996). In addition, the N-terminal CyP-homologous domain of a 150-kD tumor recognition protein, MTFK, and the FkpA from E. coli were found to not only be isomerase but also to function as chaperones during in vitro protein folding (Rinfret et al. 1994; Furutani et al. 2000; Ramm and Pluckthun 2000

Protein aggregation can occur during protein folding in vivo as well as in vitro. Correct protein folding depends on such conditions as protein concentration, pH, temperature, ionic strength, and redox environment. There is kinetic competition between the correctly folded and misfolded forms, which may or may not cause the formation of aggregates (Jeannine 1996). In addition, during the in vitro refolding of a mixture of two proteins, molecular chaperones are able to bind folding intermediates and thus block aggregation of the target protein and assist correct folding.

CK is an ideal model for studying unfolding and refolding for three reasons. (1) CK is a dimer consisting of two identical subunits, and its structure was recently resolved at 2.35 Å resolution by X-ray diffraction methods (Rao et al. 1998). (2) Extensively denatured or modified CK can spontaneously refold to its native conformation in vitro (Bickerstaff et al. 1980; Hou et al. 1983; Grossman 1984; Zhou and Tsou 1986). (3) Because CK is a large dimeric protein, its refolding is much more complicated than molecules that form small dimers or monomers, and involves several intermediates (Zhou and Tsou 1986; Wang et al. 1995; Yang et al. 1999). The competition between protein aggregation and correct folding has been previously investigated using CK (Webb et al. 1997; Zhou et al. 1997).

This paper presents a study of the effects of inactive, active, and alkylated PPIase on the CK refolding process.

Purified PPIase was stored at 4°C for use within 2 months or at −70°C for use within 6 months (Veeraraghavan and Nail 1994). Enzyme activity measurement verified that purified PPIase stored at −20°C for 2 years became inactive. PPIase did not show any prolyl isomerization activity with increasing PPIase concentrations. The conformation of inactive PPIase is similar to that of active PPIase. SEC results showed that the inactive form of PPIase was not degraded. The loss of PPIase isomerase activity was not due to structure change and polypeptide chain degradation. The essential groups for prolyl isomerase activity are His126 and Arg55 (Kallen et al. 1991). The sulfhydryl group is not necessarily essential for PPIase to exhibit the enzymatic activity (Liu et al. 1990). Although the sulfhydryl groups were also partially oxidized, His126 oxidation may have caused inactivation of PPIase stored at −20°C for 2 years.

During the CK reactivation process, the CK refolding yield was related to the protein concentration with the relative activity of 1.1 μM CK approximately 70% of native enzyme activity. However, the CK reactivation yield was about 60% of the native enzyme activity when the CK concentration was 2 μM. Increasing protein concentrations resulted in reduced CK refolding yield. In addition, PPIase affected CK reactivation in a concentration-dependent manner. Low concentrations of PPIase improved reactivation yields, with high PPIase concentrations reducing the CK yield. The data suggested that PPIase binding of folding intermediates improved the CK refolding yield, indicating that PPIase played a chaperone-like role during the CK reactivation. For high concentrations of PPIase, the lower refolding yields are possibly due to the PPIase binding early folding intermediates, and thus blocking the interaction between the enzyme and the substrate. In addition, the effect of inactive PPIase was weaker than that of active PPIase during CK reactivation. Although the two kinds of PPIase exhibited chaperone-like behavior during CK refolding, the active PPIase may more effectively bind early folding intermediates than inactive PPIase. Furthermore, the reactivation yields were both increased approximately 20% compared to the reactivation yield for self-reactivated CK when the inactive or active PPIase concentrations were four times the CK concentration. Molecular chaperones typically increase the yield but not the rate of folding reactions (Hartl 1996). Therefore, the improved CK reactivation yields were mainly due to the PPIase chaperone function and not due to its isomerization activity. The results also suggest that the chaperone function of PPIase was independent of the isomerase.

For in vitro CK folding, completely unfolded CK either correctly folds and forms an active dimer or produces misfolded intermediates and aggregates (side reactions) according to the CK folding pathway proposed by Wang et al. (1995). The CK aggregation was described as a nonspecific association through hydrophobic interactions of partially folded polypeptide chains.

In the current study, inactive and active PPIase both suppressed CK aggregation in a concentration-dependent manner, with inactive PPIase having less effect on the CK aggregation than active PPIase. An inactive dimeric folding intermediate detected in a recent study (Li et al. 2001) was shown to strongly bind with the molecular chaperone GroEL. These results suggest that inactive or active PPIase possibly bound the CK refolding intermediates as GroEL did, thus suppressing CK aggregation. This is consistent with the proposed chaperone mechanism that the chaperons work by interacting with partially folded intermediates in the folding process to prevent their aggregation and to assist correct folding. In addition, alkylated PPIase did not inhibit CK aggregation. As shown in Figure 2 ▶, the structure of alkylated PPIase is similar to that of active PPIase. Therefore, alkylated PPIase losing its ability to prevent aggregation was not due to structure change. The results indicate that cysteine residues served as peptide binding sites and as essential groups for the chaperone function of PPIase. The ability of inactive PPIase to prevent CK aggregation was weaker than that of active PPIase, probably because the cycteine residues of inactive PPIase were partially oxidized in long-term storage, thus decreasing the ability of PPIase to bind the folding intermediates of CK. This may be the main reason why the active PPIase more strongly affected the CK reactivation than the inactive PPIase.

A possible CK refolding model was proposed by Wang et al. (1995). Yang et al. (1997) showed that PPIase accelerated the slow-phase CK refolding reaction; therefore, prolyl peptide bond isomerization may be one of the rate-limiting steps. Furthermore, Yang et al. (1999) verified the existence of a monomeric intermediate in the CK refolding pathway. The present results were combined with those of previous studies (Wang et al. 1995; Yang et al. 1997, 1999), to formulate Scheme 1 as a model of PPIase assisting CK correct folding.

graphic file with name e234601.jpg

where U is an extensively unfolded CK subunit, U` is an inactive monomeric folding intermediate, U2 is an inactive dimeric folding intermediate, U2` is a misfolded dimer, N2` is a partially active dimeric folding intermediate, and N2 is an active dimer. k1 and k2 are first-order rate constants for the fast and slow phases, respectively. The final reactivation step includes adjustments of the active site conformation. Scheme 1 not only describes the CK refolding model and the chaperone and isomerase function of PPIase during the folding of dimeric CK, but also supports Freskgard's viewpoint that PPIase assisted monomeric HCA II correct folding by its chaperone and isomerase activity (Freskgard et al. 1992).

Materials and methods

Materials

Peptidyl-prolyl cis-trans isomerase was prepared from pig kidney, as described by Fischer et al. (1984). The preparation of rabbit muscle creatine kinase was as described by Yao et al. (1984). Purified PPIase and CK were homogeneous on SDS polyacrylamide gel electrophoresis. PPIase concentration was determined using the molar extinction coefficient ɛ280 = 8730 M−1 cm−1 calculated on the basis of the aromatic amino acid content (Gill et al. 1989). CK concentrations were determined using the absorption coefficient A1%1cm = 8.8 (Yao et al. 1984). Ultrapure GdmCl, Tris, chymotrypsin, N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and ATP were from Sigma. All other reagents were local products of analytical grade.

Peptidyl prolyl cis-trans isomerase assay

PPIase was determined in a coupled assay with chymotrypsin, using the synthetic short peptide N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide as described previously (Fischer et al. 1984). The PPIase was stored at −20°C for about 2 years. Fifty microliters of PPIase (16 or 100 μM) was preincubated with 50 μL chymotrypsin (5 mg/mL) in 290 μL of 50 mM Tris-HCl buffer (pH7.5) for 10 min, then 10 μL of the substrate (1 mg/mL) was added to the reaction system. Active PPIase (5 μM) was used as the positive control. PPIase activity was assayed over time by measuring the absorption at 390 nm with a Perkin-Elmer Lambda Bio U/V spectrophotometer. The temperature was kept constant at 10°C throughout the assay.

The intrinsic fluorescence emission spectra and SEC of inactive and alkylated PPIase

The inactive, active, and alkylated PPIase concentrations were all 20 μM. An excitation wavelength of 280 nm was used to measure the fluorescence intensity. The intrinsic fluorescence emission spectra were measured with a F-2500 fluorescence spectrophotometer. The reactions all occurred at 25°C.

Inactive PPIase was evaluated using SEC with superdex HR200. Active PPIase was used as the control. The SEC was recorded with an AKTA purifier.

Reactivation of CK in the presence of PPIase

The CK activity was measured using the pH-colorimetric method (Yao et al. 1984). The CK was denatured with 3 M GdmCl for 1 h. The enzymatic activity was measured in aliquots taken at suitable time intervals after the unfolded CK was diluted into the reactivation buffer in the absence and presence of PPIase. The recovery of enzymatic activity was monitored by following the absorption at 597 nm with a Perkin-Elmer Lambda Bio U/V spectrophotometer. The reactions all occurred at 25°C.

Modification of PPIase

PPIase (234 μM) was reduced by incubation with DTT (4 mM) in 30 mM Tris-HCl buffer (pH 8.0) at 25°C for 30 min and then modified by adding iodoacetic acid to a final concentration of 25 mM at 25°C for 2 h with stirring. During the modification with iodoacetic acid, the pH of the reaction mixture was readjusted to 8.0 with 0.5 M NaOH. The reaction mixture was passed through a HiTrap 5 mL-desalting column pre-equilibrated with the same buffer to remove the excess iodoacetic acid.

CK aggregation in the presence of different PPIase

GdmCl (3 M) denatured CK (150 μM) was added to a buffer (30 mM Tris-HCl, 1 mM EDTA, pH 8.0) and pre-equilibrated to 37°C in the absence or presence of various amounts of inactive PPIase, active PPIase, or alkylated PPIase, as indicated in the figure legends. The CK aggregation was followed by monitoring the turbidity at 400 nm with a Perkin-Elmer Lambda Bio U/V spectrophotometer.

Acknowledgments

This research was supported by the National Key Basic Research Specific Foundation of China No. G 1999075607.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

Abbreviations

  • CK, creatine kinase

  • CsA, cyclosporin A

  • CyP, cyclophilin

  • GdmCl, guanidine HCl

  • PPIase, peptidyl-prolyl cis-trans isomerase

  • SEC, size-exclusion chromatography

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.23301.

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