In Vitro Refolding of Human Proinsulin

KINETIC INTERMEDIATES, PUTATIVE DISULFIDE-FORMING PATHWAY, FOLDING INITIATION SITE, AND POTENTIAL ROLE OF C-PEPTIDE IN FOLDING PROCESS*

Zhi-Song QiaoDagger , Cheng-Yin MinDagger , Qing-Xin Hua§, Michael A. Weiss§, and You-Min FengDagger

From the Dagger  Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People's Republic of China and the § Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44016

Received for publication, January 28, 2003, and in revised form, March 3, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human insulin is a double-chain peptide that is synthesized in vivo as a single-chain human proinsulin (HPI). We have investigated the disulfide-forming pathway of a single-chain porcine insulin precursor (PIP). Here we further studied the folding pathway of HPI in vitro. While the oxidized refolding process of HPI was quenched, four obvious intermediates (namely P1, P2, P3, and P4, respectively) with three disulfide bridges were isolated and characterized. Contrary to the folding pathway of PIP, no intermediates with one- or two-disulfide bonds could be captured under different refolding conditions. CD analysis showed that P1, P2, and P3 retained partially structural conformations, whereas P4 contained little secondary structure. Based on the time-dependent distribution, disulfide pair analysis, and disulfide-reshuffling process of the intermediates, we have proposed that the folding pathway of HPI is significantly different from that of PIP. These differences reveal that the C-peptide not only facilitates the folding of HPI but also governs its kinetic folding pathway of HPI. Detailed analysis of the molecular folding process reveals that there are some similar folding mechanisms between PIP and HPI. These similarities imply that the initiation site for the folding of PIP/HPI may reside in the central alpha -helix of the B-chain. The formation of disulfide A20-B19 may guide the transfer of the folding information from the B-chain template to the unstructured A-chain. Furthermore, the implications of this in vitro refolding study on the in vivo folding process of HPI have been discussed.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin is a protein hormone consisting of an acid A-chain of 21 residues and a basic B-chain of 30 amino acids (1). The three-dimensional structure of insulin has been thoroughly studied by x-ray crystallography (2, 3) and NMR spectroscopy (4-6), showed that the insulin monomer is a compact globule structure with a hydrophobic core. The A-chain contains two alpha -helices (IleA2-ThrA8 and LeuA13-TyrA19, designated helix 2 and helix 3, respectively) and the B-chain consists of a central alpha -helix (SerB9-CysB19, designated helix 1), flanking turns, and extended N- and C-terminal segments. In the native structure, the N terminus of the A-chain is closely proximate to the C terminus of the B-chain. A and B chain are tethered by two interchain disulfide bridges (A7-B7 and A20-B19). The third disulfide bridge (A6-A11) is intra-A-chain disulfide. The three disulfide bonds are important in maintaining the native conformation and biological activities of the insulin molecular. The contributions of these bridges to the structure, stability, and activity of hormone have been investigated in analogues lacking selected disulfide bridges (7-12). Internal cystine A6-A11 is close to the hydrophobic core and its substitution with Ser or Ala resulted in the unfolding of helix 2 (10, 11). The interchain disulfide linkage A7-B7 is fully exposed on the surface, and its substitution with Ser leads to a marked decrease in the thermodynamic stability and extent of folding as compared with that of the substitution of internal cystine A6-A11 (7, 13). NMR studies showed that all of helix 2 and part of helix 3 were unstructured in [SerA7,SerB7]DKP insulin and therefore resulted in a looser conformation of the overall molecular (7). Whereas removal of A7-B7 or A6-A11 results in only segmental unfolding, cystine A20-B19 appears to be integral to the overall structure and necessary for the biosynthetic expression (13). Disulfide isomers retaining this core disulfide bridge exhibit native-like partial folds with nonnegligible biological activity. Such isomers provide examples of kinetic traps in an energy landscape (7-9, 14).

Although the functional form of human insulin is double-chain, human insulin was synthesized in vivo as a single-chain preproinsulin with a signal peptide at the N terminus of the B-chain and a connecting peptide between the B- and A-chain (15, 16). Following cleavage of an N-terminal signal sequence in the endoplasmic reticulum, the nascent polypeptide folds and is packaged into secretary granules as a proinsulin. Its C terminus of B-chain connects the N terminus of A-chain by the connecting peptide consisting of two bibasic amino acids at each end of the C-peptide of 31 amino acids (17, 18). The amino acid sequence of human proinsulin (HPI)1 is shown in Fig. 1. Processed by a specific set of protease, proinsulin is converted into insulin and C-peptide in the B-cell granule (19, 20). Due to the flexibility of the C-peptide, there is still no report of the crystal or solution structure of the proinsulin, although proinsulin could be co-crystallized in a 1:1 complex with insulin (21, 22). Models of HPI structure were proposed in the 1970s, suggesting that the flexible C-peptide runs across the surface of A-chain (22, 23). Weiss et al. (24) also showed by comparative 1H NMR and photochemical dynamic nuclear polarization studies that the insulin moiety of proinsulin is similar to insulin and that the connecting peptide is largely unstructured.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1.   Amino acid sequence and disulfide linkage pattern of HPI. Amino acids are shown in one-letter code, and cysteine residues are shown as dark solid circles. For convenience of description, the nomenclature of the amino acids in HPI refers to that of insulin when related to the A- and B-chain. The arrows indicate the potential cleavage site of endoproteinase V8. Peptide fragments obtained after enzyme cleavage are designated as F1-F9, assuming that the disulfide bonds are reduced.

Since the discovery by Anfisen in 1960s (25) that the folding information of proteins resides in the primary amino acid sequences, great efforts have been devoted to the study of the molecular mechanism of protein folding. Two important questions in the mechanism of protein folding are still not clear: (i) how the folding is initiated and (ii) what pathway it follows. For the latter question, one can characterize the intermediates appearing in the folding process (26, 27). Disulfide linkages are always important factors in maintaining the native conformation of many single-domain globule proteins containing disulfide bonds, and their pairings are always coupled to the formation of the functional structure during the oxidized refolding of such proteins (28, 29). Thus, the formation of disulfide bonds could be used as a useful tool in probing the protein-folding pathway (30). The folding pathways of many proteins, such as bovine pancreatic trypsin inhibitor (31-35), ribonuclease A (36-38), epidermal growth factor (39, 40) have been widely studied by using this method. Several members of the insulin superfamily have been identified, including insulin, IGF-I (41), IGF-II (42), relaxin HI (43), amphioxus insulin-like peptide (44), prothoracicotrophic hormone-II (45), molluscan insulin-related peptide (46), Caenorhabditis elegans insulin-like peptide (47), etc. Among these members, only the single-chain IGF-I has been extensively investigated into its disulfide-forming pathway (48-53). Although insulin is one of the best characterized proteins in their structure and function, its in vitro disulfide folding pathway remains far from understood due to the difficulties brought by its double-chain structure. Early studies by Steiner et al. (54) have demonstrated that proinsulin exhibits the expected property of spontaneously reforming its native structure in alkaline solution after complete reduction in urea, which supported the view that the single-chain proinsulin is the biosynthetic precursor of insulin. We recently studied the disulfide-forming pathway of a single-chain porcine insulin precursor (PIP) and suggested that reduced PIP proceeds through two pathways to reach the native PIP (55). Since insulin is synthesized and folded as a single chain proinsulin in vivo (16), we further studied the in vitro disulfide-forming pathway of human proinsulin. The results have shown that there are some differences in the folding behavior between HPI and PIP, indicating that C-peptide has a profound impact on the molecular mechanism of HPI refolding. The studies on the refolding process of the captured intermediates of HPI, on the other hand, show that some fundamental similarities exist in both folding mechanism of HPI and PIP. We demonstrate that the length of the connecting peptide has a profound effect on the mechanism of folding. Whereas PIP folds in distinct steps via one- and two-disulfide intermediates (55), HPI is first caught in kinetic traps, forming at least four nonnative disulfide isomers, which then rearrange to the native state.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Recombinant HPI with purity greater than 98% was kindly provided by Lilly and confirmed by HPLC on a reverse phase C8 column. Protein concentration of HPI, reduced/denatured HPI, and all of the intermediates was quantified by a spectrometer and calculated with the absorption constant A278 (1 cm, 1.0 mg/ml) = 0.65 according to Ref. 56. Endoproteinase Lys-C and V8 were of sequencing grade and purchased from Sigma. The snakeskin dialysis tube with a molecular mass cut-off of 3,500 daltons was the product of Pierce. IAA, GSH, and GSSG were ultrapure and obtained from Amerresco. Ultrapure dithiothreitol and L-Arg were purchased from Sigma. Ultrapure urea and guanidine-HCl were the products of Promega. Acetonitrile and trifluoroacetic acid were of HPLC grade. All of the other reagents used in the experiment were of analytical grade.

Refolding of Fully Reduced/Denatured HPI (frdHPI)-- Purified HPI was dissolved in 100 mM Tris-HCl buffer (pH 8.7), containing 8 M guanidine-HCl, 1 mM EDTA, and 100 mM dithiothreitol. Reduction of the protein was carried out at 37 °C for 30 min. Thereafter, the buffer was immediately exchanged to 10 mM HCl by gel filtration using Sephadex G-25 (Amersham Biosciences) and stored at -80 °C for later use. The refolding of reduced and denatured protein was initiated by diluting the frdHPI with the refolding buffer (100 mM Tris-HCl, 5 mM EDTA, different concentrations of GSH and GSSG, with or without L-Arg) at a final protein concentration of 0.1 mg/ml. The refolding reaction was carried out at 16 °C for 16 h and quenched by adding trifluoroacetic acid to pH 1.0, and then the mixture was analyzed on HPLC using a C4 reverse phase column to quantify the refolding yield. The impacts of different redox constitutions, pH, and L-Arg on the refolding yield of HPI have been tested, and an optimized refolding condition with the best yield of HPI was selected to carry out the in vitro studies of the disulfide-refolding pathway of HPI.

Trapping, Separation, and Purification of Refolding Intermediates-- Refolding of frdHPI was initiated by adding frdHPI in the prewarmed (16 °C) refolding solution (100 mM Tris, pH 10.0, 5 mM EDTA, 1 mM GSH, 5 mM GSSG) at a final concentration of 0.1 mg/ml. The refolding process was quenched by acid or iodoacetic acid in a time course manner. For the acid quenching method, aliquots of protein solution were removed at different refolding times and quickly mixed with an equal volume of 2% trifluoroacetic acid to lower the pH to 1.0. For the iodoacetic acid method, the refolding reaction was quenched by adding one-fourth volume of freshly prepared 0.5 M IAA in 50 mM Tris-HCl (pH 8.5), and the carboxymethylation reaction was performed at room temperature for 5 min. To observe the temporal distribution of refolding intermediates, the IAA-quenched solution was adjusted to pH 1.0 with trifluoroacetic acid and immediately analyzed by HPLC. For large scale preparation of the trapped intermediates, the solution containing IAA-trapped intermediates was dialyzed against 50 mM NH4HCO3 (pH 8.0) at 4 °C and thereafter lyophilized. Then the refolding mixture was separated by HPLC on a reverse phase C4 (Sephasil peptide, ST 4.6/250 mm; Amersham Biosciences) column. Unless otherwise indicated, the solvent A used in HPLC was water containing 0.15% trifluoroacetic acid, and the solvent B was 60% acetonitrile containing 0.125% trifluoroacetic acid. The linear elution gradient was 40-80% of solvent B in 30 min with a flow rate of 0.5 ml/ml, and the monitoring wavelength was 280 nm. The corresponding peaks were collected manually and lyophilized. Then the partially separated intermediates were further purified by a semiprepared C8 (Sephasil peptide, ST 4.6/250 mm; Amersham Biosciences) column and characterized by an analytical C8 column (ZORBAX SB, 4.6/150 mm; DuPont) on reverse-phase HPLC.

Disulfide Linkage Analysis of the Intermediates by Enzyme Digestion-- Two endoproteinases, Lys-C and V8, were utilized to digest the intermediates of HPI in order to characterize their disulfide linkages. Lys-C and V8 cleave at the carboxyl terminus of Lys and Glu, respectively. There are two lysine residues and eight glutamic acids in the amino acid sequence of HPI. In general for the enzyme digestion, 10 µg of the intermediates was dissolved in 10 µl 100 mM NH4HCO3 (pH 8.0), and 1.5 × 10-3 units of Lys-C or 0.5 µg of V8 was added. HPI was used as a positive control in each of the enzyme digestion. The reaction was carried out at 25 °C for 16 h and quenched by adding 90 µl of 0.3% trifluoroacetic acid. Then the digestion mixture was immediately analyzed by reverse-phase HPLC on C8 column (ZORBAX SB-C8, 5µ, 4.6/150 mm; DuPont). The elution gradient was 25-65% of solvent B linear in 35 min. The flow rate was 0.5 ml/min, and the detector wavelength was set at 210 nm.

Folding of the Intermediates-- Isolated intermediates of HPI with three isomeric disulfide bridges were dissolved in 5 mM HCl, and the protein concentration was adjusted to 0.2 mg/ml. Before the folding of the intermediates, protein solution and a 2-fold concentration of refolding buffer (200 mM Tris, pH 10.0, 10 mM EDTA, 2 mM GSH, 10 mM GSSG) were incubated at 4 °C for 10 min, respectively. The folding reaction was initiated by mixing them with equal volume and carried out at 4 °C. Aliquots of the folding solution were removed in a time course manner and mixed with an equal volume of 2% trifluoroacetic acid to stop the folding process. Then the mixture was analyzed by reverse-phase HPLC on a C4 column (Sephasil peptide, ST 4.6/250 mm; Amersham Biosciences) with a linear gradient of 50-80% of solvent B in 30 min. The flow rate was 0.5 ml/min, and the detector wavelength was 230 nm.

Circular Dichroism Studies-- All of the samples were dissolved in 10 mM HCl except for the IAA-modified frdHPI in 50 mM NH4HCO3 (pH 8.0). The protein concentration was all adjusted to 0.25 mg/ml by UV absorption at 278 nm. Circular dichroism measurements were performed on a Jasco-700 circular dichroism spectropolarimeter at 25 °C. For the far-UV CD spectra, samples were scanned from 190 to 250 nm and accumulated twice at a resolution of 1.0 nm with a scanning speed of 50 nm/min. The cell length was 0.1 cm, and the stepwise increase was 0.1 nm. For the near-UV CD spectra, samples were scanned from 250 to 310 nm and accumulated twice at a resolution of 1.0 nm with a speed of 50 nm/min. The cell length was 1.0 cm. All of the CD data were expressed as mean residue ellipticity. The secondary structure contents of the samples were estimated by using the software J-700 for Windows Secondary Structure Estimation, Version 1.10.00 (JASCO Corp.), equipped with the Jasco-700.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In Vitro Refolding of HPI with High Yield-- To optimize the refolding conditions, the concentration of GSH and GSSG, the pH, and the absence or presence of the L-Arg have been tested. After a series of tests, we have found that several factors in the optimized refolding condition of HPI are obviously different from those of PIP, although the difference of two molecules in sequence is only the length of connecting peptide. First, L-Arg was not absolutely required for the refolding of HPI, whereas the yield of PIP was lower than 15% in the absence of L-Arg. Even oxidized by air, HPI could fold spontaneously with a high yield up to 50%. Thus, it is clear that HPI refolds more efficiently than PIP in vitro. Second, predominance of GSSG in the redox buffer is required for the high yield of HPI, whereas GSH is required for PIP. Table I shows the recovery of refolded native HPI reached as high as 84% when the frdHPI was diluted in the refolding buffer containing 100 mM Tris, pH 10.0, 5 mM EDTA, 1 mM GSH, and 5 mM GSSG at a final concentration of 0.1 mg/ml and incubated at 16 °C for 16 h. Thus, the optimum was used in the in vitro refolding study of HPI.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Refolding yield of HPI under different oxidized conditions
The refolding percentage was calculated from the peak area integration of the refolded HPI on HPLC with the frdHPI used for the refolding as a reference. The data should allow an S. D. value of ± 5%. Unless otherwise indicated, the refolding buffer contains 100 mM Tris, 5 mM EDTA, and the indicated concentration of GSH and GSSG. The protein concentration of HPI was 0.1 mg/ml, and the refolding reaction was carried out at 16 °C for 16 h.

Temporal Distribution of the Disulfide Intermediates of HPI-- After the refolding process was initiated by adding frdHPI into the refolding buffer, the reaction was quenched at different time points by lowering the pH to 1.0 with trifluoroacetic acid, and the mixture was analyzed immediately by reverse-phase HPLC on a C4 column. The temporal distribution of the refolding intermediate species is shown in Fig. 2. Four obvious intermediates, designated by elution time, P1, P2, P3, and P4, were observed during the refolding process of HPI. In the first 2 min of the refolding, only a very small proportion of the intermediates as well as the frdHPI can be identified by HPLC. The SDS-PAGE results (data not shown) of the deposits and the supernatants of the acid-quenched refolding mixture showed that intermediates during this period were easily acid-precipitated. Analysis of the precipitate of the refolding mixture at different time points by SDS-PAGE (data not shown) showed that they were mostly disulfide-linked intermolecular aggregation. These aggregated species always act as off-pathway intermediates and hence prevent the HPI refolding efficiency to reach a full scale. After 2 min of the refolding process, HPI and the four intermediates begin to dominate the folding intermediate species. Fig. 3A shows the recovery curve of the native HPI calculated from the HPLC chromatography, and Fig. 3B shows the time-dependent distribution of the four intermediates. The contents of all of the intermediates came to a climax at 6 min, and then the content of P1 decreased slowly, whereas P2 and P4 decreased more quickly. Although there are some minor intermediates other than P1, P2, P3, and P4 at 2 min of the folding, it is very difficult to identify them due to the lower contents.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2.   Temporal distribution of the intermediates trapped by trifluoroacetic acid (A) and IAA (B) at different time points during the oxidized refolding process of HPI in vitro. The time that the refolding reaction was quenched is indicated at the right bottom of each chromatogram. The four peaks designated as P1, P2, P3, and P4 are indicated at 4 min of acid-quenched reaction. The three peaks designated as IA1, IA2, and IA3 are indicated at 6 min of IAA-quenched reaction. The linear elution grade was 40-80% solvent B in 30 min with a flow rate of 0.5 ml/min, and the wavelength of the monitor was set at 230 nm.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   Time-dependent recovery of native HPI (A) and the refolding intermediates (B). The percentage of HPI refolding was calculated from the peak area integration on HPLC relative to the reduced HPI used for the refolding. The refolding percentage at 16 h was not indicated in this curve, since there is little difference from that at 90 min. The yield of intermediates was expressed as the percentage of the total reduced HPI used for the refolding.

The refolding process was also quenched by IAA and observed by HPLC as shown in the right lane of Fig. 2. In contrast with the acid-quenched results, there are only three major intermediates peaks, designated as IA1, IA2, and IA3, respectively, along the refolding. The elution time of IA1 and IA2 is the same as that of acid-trapped P1 and P2. IA3 was later confirmed by native electrophoresis to be a mixture of acid-trapped P3 and P4. The lower resolution of HPLC in IAA-trapping than acid-trapping was probably caused by IAA.

Populated Intermediates Are Disulfide Isomers-- The refolding process was quenched by trifluoroacetic acid at 6 min, and the mixture was separated by HPLC on a C4 column. The partially separated intermediates, P1, P2, P3, and P4, were modified by IAA and purified by HPLC on a C8 column. Then the molecular mass of the purified IAA-modified intermediates was measured by ESI-MS, and all of them were shown to be 9388, which was identical to that of native HPI. This indicates that all of the intermediates have no reaction with IAA and that P1, P2, P3, and P4 contain three intact disulfide bonds, with at least two nonnative ones. Therefore, the four intermediates are scrambled disulfide isomers of HPI. These purified intermediates were further analyzed by pH 8.3 native electrophoresis, as shown in Fig. 4. P1, P2, and P3 were a little slower than native HPI in the electrophoresis, indicating that they have a looser conformation than HPI. P4 was much slower than native HPI and the other intermediates, so it has a much more flexible conformation. Although the purified intermediates were symmetrical peaks on HPLC profiles as shown later, there are some bands at the lower portion of each lane of the intermediates on pH 8.3 native electrophoresis. These minor bands were probably the results of deamidation of the intermediates, which was observed in our previous result (55).


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 4.   15% native PAGE of the purified intermediates. Lanes 1 and 7, native HPI and IAA-modified reduced and denatured HPI. Lanes 2, 3, 4, and 6, the purified intermediates P1, P2, P3, and P4, respectively. Lane 5, the proteins from peak IA3 in Fig. 2 and a mixture of P3 and P4. The additional minor bands indicated by an asterisk are the deamidated products of each intermediate caused by acid during the purification. Each intermediate shows an asymmetric peak on HPLC profiles and single band on SDS-PAGE.

For large scale preparation of the four intermediates, the refolding process was stopped by adding IAA that will modify all of the free thiols. Then the mixture was dialyzed, lyophilized, desalted, and separated by RP-HPLC on a C4 column. Because the four intermediates are close in the elution profile, it is very difficult to thoroughly separate them by HPLC once. So, we further purified them with two rounds of RP-HPLC on a semiprepared C8 column. The purity and elution time were identified by RP-HPLC on an analytical C8 column.

Disulfide-linkage Elucidation of the Four Intermediates-- Four intermediates were first digested by endoproteinase Lys-C, which cleaves at the carboxyl terminus of lysine residues (B29 and C34), and analyzed by RP-HPLC on a C8 column as shown in Fig. 5. All of the major peaks of the mixture of the enzyme-digested intermediates on HPLC were collected, and their molecular weight was measured by ESI-MS as shown in Table II. The left peaks of each profile in Fig. 5 (HPI-Fa, P1-Fa, P2-Fa, P3-Fa, and P4-Fa) correspond to the fragment containing the C-peptide. The molecular weights of another two peaks in Lys-C-digested P4, peaks P4-Fb and P4-Fc, correspond to the fragments of A- and B-chain with intrachain disulfide bridges, respectively. This indicates that P4 should have a nonnative B9-B19 disulfide bond and no inter-AB disulfide linkages. Combined with the results from Fig. 4, P4 is the most flexible isomer with a B7-B19 intra-B-chain bridge. Lys-C-digested P1-P3 in Fig. 5 showed the same molecular weight of 5862 for peaks P1-Fb, P2-Fb, and P3-Fb, implying that P1-P3 are three disulfide isomers with two interchain disulfide bridges.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 5.   HPLC chromatograms of the peptide mapping of HPI and intermediates digested with Lys-C. Chromatograms A-E show the Lys-C-digested HPI, P1, P2, P3, and P4, respectively. The elution conditions are described under "Experimental Procedures." The absorbance was measured at 210 nm using a Gilson 115 UV detector. Chromatograms A-E represent the Lys-C-digested HPI, P1, P2, P3, and P4, respectively. Peaks on the left portion of the each chromatogram, including HPI-Fa, P1-Fa, P2-Fa, P3-Fa, and P4-Fa, have the same elution time and correspond to the major connecting peptide, which was verified by the mass spectrum data in Table II. Peak P4-Fb and P4-Fc in P4 correspond to the fragments of B-chain and A-chain, respectively, with intrachain disulfide bonds. Peaks P2-Fb and P3-Fb and the major peak in P1 have different elution times on HPLC but the same molecular weight corresponding to the A- and B-chain linked by interchain disulfide bonds. Although the contents of all other minor peaks in P1 are too low to be purified and identified by mass spectrum, some of the peaks have the same elution time as P2-Fb and P3-Fb. This indicates that P1 may be a mixture of several disulfide isomers.


                              
View this table:
[in this window]
[in a new window]
 
Table II
Molecular mass of the peptide fragments of samples digested by endoproteinase Lys-C in Fig. 5

To further elucidate the disulfide linkage of the four intermediates, endoproteinase V8 that cleaves at the carboxyl terminus of Glu residues was utilized to digest these intermediates. Because native HPI contains eight Glu residues and two inter-AB disulfide bonds (A7-B7 and A20-B19) each link two pieces of fragments as indicated in Fig. 1, only seven fragments would be obtained after V8 digestion. After the enzyme digestion, the mixture was separated by HPLC, and each profile is shown in Fig. 6. The major peaks were collected, and their molecular weight was measured by ESI-MS as shown in Table III. From mass values shown in Table III, the corresponding fragment of each peak on HPLC could be identified. Peaks a', a1', a2', a3', and a4' in Fig. 6 correspond to fragment F5 in Fig. 1. In peak a, a1-a4 correspond to F7. In peak b, b1-b4 belong to F3, and in peak d, d1-d4 are F6. All of these four peaks (a', a, b, and d) correspond to the segments without disulfide bonds. V8-digested P1 showed these four peaks and other small peaks, which are similar to c2, e2, f3, and f4 in three bottom profiles in Fig. 6. This suggests that P1 may be a mixture of several intermediates with interchain disulfide bonds. The molecular weights of peaks c2 and e2 of V8-digested P2 are identical to those of peak c and e of V8-digested HPI, respectively, but the retention time of peak e2 is shorter than that of peak e of HPI. This demonstrates that P2 contains the disulfide bond A20-B19 and another nonnative inter-AB one, possible B7-A6 or B7-A11. For the V8-digested P3, the major peak f3 with a molecular weight of 1993 corresponds to that of fragment F1 linked with F9 of HPI. Accordingly, it could be expected that P3 contains a nonnative B7-A20 disulfide linkage. For the V8-digested P4, in addition to peak a4, b4, and d4 as described above, peak f4 has a molecular weight of 2347 that was the sum of the molecular weight of fragments F1 and F2 in Fig. 1. This indicates that P4 contains the intra-B-chain disulfide B7-B19. Due to the absence of amino acid sequencing of the intermediates, we could not distinguish the disulfide linkage patterns among A6, A7, and A11. All of the possible disulfide linkages of intermediates P2-P4 are shown in Fig. 7. For intermediates P3 and P4, they may be one of these three possible disulfide linkages or the mixture of them. The disulfide linkage pattern of P2 has two possibilities, among which isomer a and b correspond to the insulin isomer swap and swap-2, respectively (57, 67). There are a total of 14 possibilities of the disulfide isomers of HPI, and P1 may be a mixture of all or part of the other isomers except for P2, P3, and P4.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 6.   HPLC chromatograms of the peptide mapping of HPI and intermediates digested with endoproteinase V8. The elution and detection system are the same as that in Fig. 5. Chromatograms A-E represent the V8-digested HPI, P1, P2, P3, and P4, respectively. For the V8-digested native HPI, there are a total of six peaks, among which peaks a, a', b, and d correspond to the peptide fragments (F5, F7, F3, and F6 in Fig. 1, respectively) that do not contain cystines, so these four peaks will consistently appear in V8-digested products of all intermediates. Peaks c and e in HPI correspond to the disulfide-linked fragments F2 + F9 and F1 + F8 respectively, so these two peaks may vary between different intermediates that contain different disulfide linkage patterns. The mass spectra results for these peaks are listed in Table III.


                              
View this table:
[in this window]
[in a new window]
 
Table III
Molecular mass of the peptide fragments of samples digested by endoproteinase V8 in Fig. 6
The peaks in the intermediates corresponding to peaks a, a', b, and d in HPI were not measured because they have the same molecular weight as those of HPI.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 7.   Schematic representation of the possible disulfide linkages of the HPI folding intermediates. The disulfide bonds that have been verified by mass spectrum are indicated by a solid line. Those unclarified disulfide bonds were indicated as a dotted line. Intermediate P1 was not shown here and it may be a mixture of disulfide isomers of HPI except for P2, P3, and P4. The disulfide linkage pattern of isomers a and b in P2 correspond to the insulin isomer swap and swap-2, respectively.

Intermediates are Partially Folded-- The far-UV CD spectra of the four intermediates are shown in Fig. 8A. The results show that there is obvious reduction in alpha -helix content of all these intermediates relative to native HPI. Calculated from the CD spectra of each sample, P1-, P2-, P3-, P4-, frdHPI-, and IAA-modified frdHPI retained approximately helical contents of 40, 40, 55, 12, 22, and 0% related to total helices of native HPI, respectively. Because HPI contains 27 helical residues, the number of helical residues in P1-P4 is in turn estimated to be 11, 11, 15, and 3, respectively.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 8.   Circular dichroism spectra of the intermediates. A, far-UV CD spectra of HPI (N), frdHPI (U), and all of the intermediates. frdHPI is the fully reduced and denatured HPI in 5 mM HCl. IAA-frdHPI is the IAA-modified frdHPI in 10 mM NH4HCO3 (pH 8.0). At 222 nm, the spectra from bottom to top were as follows: HPI, P3, P1, P2, P4, frdHPI, and IAA-frdHPI, respectively. B, near-UV CD spectra of HPI (N), frdHPI (U), and intermediates P1, P3, and P4. P2 was not measured for the absence of enough samples.

The near-UV CD spectra of HPI, P1, P3, P4, and frdHPI are shown in Fig. 8B. Intermediate P2 has not been measured, because we are unable to get enough samples for this analysis. From their spectra, P4 is most similar to frdHPI, whereas P1 and P3 are much closer to HPI than P4. This indicates that P4 has the less folded conformation, whereas P1 and P3 with the interchain disulfide bonds have the partially structured conformation.

Reassortment of the Disulfide Isomeric Intermediates-- All of the purified intermediates with the three isomeric disulfide bridges were reconstituted into the refolding buffer to allow the reshuffling of the disulfide bonds to native forms. Refolding was quenched by trifluoroacetic acid and immediately analyzed on HPLC in successive times in Fig. 9, in which all four intermediates could refold into native HPI with the correct disulfide pairing. This indicates that P1, P2, P3, and P4 are on-pathway intermediates along the folding process of HPI. P2 with A20-B19 native interchain bridge provides the most routes to the native state. There are three lines of evidence. (i) The refolding speed of P2 is faster than the other three intermediates. After 15 min of refolding, more than 50% of the P2 has been converted into the native HPI. (ii) The refolding process of any intermediates is always concomitant with the appearance of the other three intermediates. However, during the refolding of P2, the proportion of the other intermediates is the lowest among all of the intermediates. (iii) P2 accumulates during the refolding process of P1, P3, and especially P4. Although P1 is closer to native HPI than P2 at elution time on HPLC, P2 is still a major intermediate during the disulfide rearrangement of P1. These observations suggest that P2 with one native disulfide bridge, A20-B19, is the most direct intermediates to reach the native HPI. P1, P3, and P4 may have to rearrange to form P2 before reaching the native HPI.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 9.   Refolding of the HPI intermediates. The intermediates were reconstituted in the same refolding buffer with the HPI, and the refolding process of the four intermediates was quenched at a fixed time point and analyzed on HPLC under the conditions described under "Experimental Procedures." A-D, refolding chromatograms of P1, P2, P3, and P4, respectively. The refolding times are indicated at the right bottom of each chromatogram. The intermediates indicated by the arrows during the refolding were identified by their elution time compared with that of the purified intermediates.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Disulfide Folding Pathway of HPI and the Possible Molecular Mechanism-- During the refolding process of HPI, only scrambled disulfide isomers with three disulfide bonds were captured as intermediates. Unlike the refolding of PIP, there are no accumulated intermediates with one or two disulfide bridges identified. It is possible that the different refolding behavior between HPI and PIP might have been caused by the different conditions, such as the excess GSSG over GSH in the redox buffer in HPI refolding we used. To check this possibility, we carried out the refolding process of HPI under the similar situation with that of PIP, in which the refolding buffer contained 50 mM Tris, pH 9.5, 0.5 M L-Arg, 5 mM EDTA, 5 mM GSH, 0.5 mM GSSG, and the protein concentration was 0.1 mg/ml. Time-dependent distribution of the intermediates was observed on HPLC, and there were still no additional detectable intermediates (data not shown). This implies that the different patterns of intermediates accumulated along the refolding process of HPI and PIP are not caused by different refolding conditions used but are determined by the inherent properties of the distinct proteins.

Of the four intermediates of HPI, those with two interchain disulfide bonds, such as P1, P2, and P3, always possess partially structured conformation, as deduced from the results of native gel electrophoresis and CD spectra. Only P4 without inter-AB disulfide bridges has a very flexible conformation, which is similar to the frdHPI. This indicates that the formation of interchain disulfide linkages, whether native or nonnative, is a crucial factor to construct the native conformation.

For the single-domain protein with disulfide bridges, the hydrophobic interaction and the covalent disulfide linkage are two important forces to maintain the native conformation and direct the folding pathway of a protein (30). The oxidative refolding pathway of HPI is also governed by the mutual interaction of the two forces. The fact that the accumulated scrambled disulfide isomers are refolding intermediates and an equilibrium among the intermediates of HPI suggests that the folding of HPI probably adopts the following mechanism. At the very beginning of the HPI refolding, where the hydrophobic interaction has not contributed to the folding of the peptide chain, the nearest free thiols on the random-coiled frdHPI paired randomly and rapidly to form the three scrambled disulfide linkages, and most of them are nonnative as the intermediates of P4. Subsequently, driven by the hydrophobic interaction, the secondary structure is gradually formed in the intermediates, which initiate and direct the reshuffling of the nonnative disulfide bonds to form the native disulfide bonds and consequently solidify the partially formed conformation. Accordingly, the intermediates, such as P1, P2, P3, and P4, with nonnative disulfide bridges were captured in the early stage of the HPI refolding.

Refolding of the four intermediates suggests that P2 possessing native disulfide bond A20-B19 is the most important intermediate during the disulfide reshuffling process of HPI, and the other three intermediates must rearrange their disulfide pairings to form P2 on their way to the native HPI. Based on the observations and deductions above, we have proposed a putative disulfide folding pathway of HPI in vitro as shown in Fig. 10. In the absence of oxidized reagent such as GSSG, free thiols of frdHPI can also pair randomly and rapidly to form in turn the one-disulfide and two-disulfide intermediates until consequently native HPI or the three-disulfide scrambled isomers such as P1, P2, P3, and P4. Thereafter, the disulfide bonds in the isomers of intermediates begin to reshuffle. P4 without the interchain disulfide linkage could convert directly, or indirectly by way of P1 or P3, into the P2 by disulfide rearrangement. P1 and P3 could interconvert each other, and both of them could convert into P2 before they finally refold into the native HPI.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 10.   Schematic representation of the putative disulfide folding pathway of HPI in vitro. The double arrow indicates the equilibrium between each intermediate species; the equilibrium has a greater tendency for the end with the bigger arrow. I-III represent the intermediate mixtures with one, two, and three disulfide bonds, respectively. R and N, frdHPI and native HPI, respectively.

NMR structure analysis of the insulin analogues lacking the disulfide bridge A7-B7 or A6-A11 showed that B-chain retains native-like supersecondary structure, whereas most of the A-chain is disordered (7, 10, 12). These observations suggest that the B subdomain provides a template to guide folding of the A-chain. Accordingly, we suggest that the disulfide reshuffling during the folding of HPI was also a process directed by the folding information of B-chain templates. The partially structured P1, P2, and P3 should have a native-like B subdomain with intact helix 1. The folding information in the preformed B-chain was transferred to the A-chain by means of C-peptide and interchain disulfide bridges, and hence the folding of the A-chain guides the disulfide reshuffling in these intermediates. For the P4 with a very felxible conformation, the intra-B chain disulfide bridge B7-B19 may be a hindrance to form the central helix B9-B19. Only after the untethering of the disulfide B7-B19, the native-like structure in the B-chain can be formed and consequently guide the folding of A-chain as a template and reshuffling of nonnative disulfide linkage.

Formation of the A20-B19 Bridge Is the Most Important Step in the Folding Pathways of Both PIP and HPI-- Some differences exist between PIP and HPI in their disulfide refolding pathways. First, only three intermediates with one or two disulfide bonds but not with three disulfide bonds were captured during the oxidized refolding of PIP (55). However, four intermediates with three disulfide bonds were identified during the oxidized refolding process of HPI. Second, most of the disulfide bonds in the PIP intermediates are native, whereas those in HPI are nonnative. Third, the rate-limiting step during the refolding of PIP resides in the conversion of the 2SS intermediate into the native PIP, whereas for HPI, the rate-limiting step was the disulfide reshuffling in the 3SS isomers.

Nevertheless, intrinsic similarities of refolding mechanism also exist between PIP and HPI. We have proposed two disulfide forming pathway of PIP during its refolding in vitro. One begins with the first formation of A6-A11, and the other begins with A20-B19 (55). Contrary to the first formation of A20-B19 requiring the long range interaction in the reduced polypeptide, disulfide linkage A6-A11 is the first favored possibly for the proximity of these cysteines in the sequence, and hence this pathway will eventually shift to the pathway in which A20-B19 first forms. Two partially structured intermediates, 2SSPIPa and 2SSPIPb, both have the disulfide bond A20-B19. Therefore, formation of A20-B19 seems to be a crucial step in the folding of PIP, because the A20-B19 disulfide bond participates in and stabilizes a molten subdomain, which is important for the initiation folding of PIP. During the refolding of HPI, P2 is the most important one among the four captured intermediates because of its faster speed and lower proportion of other intermediates during its rearrangement to HPI. All of the other three intermediates may have to reshuffle their disulfide bonds to form P2 before they can reach native HPI. Given that P2 contains a native A20-B19 disulfide bond, it is reasonable to conclude that formation of A20-B19 is an important factor along the folding pathway of HPI. The possible role of the formation of A20-B19 in the disulfide isomers is to anchor the A- and B-chain of HPI in the native-like orientation. Therefore, in the both refolding pathway of PIP and HPI, the formation of A20-B19 is a crucial step.

The pairwise substitution of internal disulfide A20-B19 with serine in PIP resulted in expression of a product that was too low to be detectable (13). This further suggests that A20-B19 bond is important in the folding pathway and maintaining its native conformation. Recently, a PIP mutant retaining only one disulfide, A20-B19, has been successfully expressed in yeast (71). CD results showed that it retained certain secondary structure, and the in vitro refolding experiment showed that CysA20 and CysB19 could pair rapidly with high yield. This peptide model of folding intermediates clearly demonstrated that A20-B19 is the most important one among the three disulfide bonds.

Folding Initiation Site of HPI/PIP-- One of the most important questions in protein folding is how the folding reaction is initiated (57). The folding rates and mechanisms appear to be largely determined by the topology of the native/folded state, whereas the initiation of protein folding always requires several residues that are located in the sequence to form a nucleation site as an initiation site of folding (58, 59). Although the structure and function of insulin have been amply documented, the folding initiation site of insulin/HPI/PIP is rarely studied.

The disulfide linkage patterns and native alpha -helix content of the partially structured intermediates of PIP and HPI are listed in Table IV, and the peptide models of the insulin folding intermediates whose structures have been studied by NMR are listed in Table V (7, 9, 10, 12). By comparing the peptide models with our captured intermediates here, we could infer that all of the partially structured intermediates of PIP and HPI retain helix 1. Therefore, it is reasonable to suggest that helix 1 is firstly formed during the folding of PIP and HPI. The formation of helix 1 in the B-chain contributes substantially to the native-like conformation of the B-chain that will guide the folding of A-chain. Thus, the folding initiation site of PIP and HPI should reside in the central helix 1 in which some conservative hydrophobic residues, such as LeuB11, ValB14, and LeuB15, could be the major components of the initiation site. The hydrophobic collapse of these residues at an early stage of folding forms the initiation site of HPI/PIP.


                              
View this table:
[in this window]
[in a new window]
 
Table IV
Helical content and disulfide linkage pattern of the intermediates captured during the oxidized refolding process of PIP and HPI


                              
View this table:
[in this window]
[in a new window]
 
Table V
The constructed peptide models of the disulfide intermediates during the oxidized refolding insulin analogues
1SS, 2SS, and 3SS represent the peptide models containing 1, 2, and 3 disulfide bonds, respectively. ND, not determined by NMR.

Potential Role of the C-peptide in the Refolding of HPI-- As shown under "Results," two major differences exist between the folding behavior of PIP and HPI. First, the in vitro refolding yield of HPI could easily come to a high level, whereas it is difficult for PIP to refold efficiently in the same condition. Second, HPI and PIP adopt different disulfide-forming pathways during the in vitro folding. Because the only difference of sequence between PIP and HPI is that the connecting peptide in which PIP is a dipeptide (Lys-Ala), whereas that of HPI is a C-peptide plus cleavage site at each end, so it is the connecting peptide (mainly the C-peptide) of HPI that was responsible for the discrepancy in folding behavior with PIP.

How the flexible C-peptide contributes to the high refolding yield of HPI? The 31-residue C-peptide contains five acidic amino acids and no basic ones, so it is a negative charge-rich peptide fragment. The concentrated charge in the peptide might result in the intermolecular repulsion during the refolding of HPI and hence attenuated the intermolecular hydrophobic interaction that always leads to the aggregation. Therefore, the acidic residues in C-peptide may account for the higher refolding yield of HPI. Our results are consistent with a recent experiment in which both alanine replacement mutation and deletion of three highly conserved acidic residues (EAED) at the N terminus of the C-peptide resulted in serious aggregation during HPI refolding (60). Therefore, it is possible that the C-peptide acts as an intramolecular chaperone in the folding of HPI. Actually, there are many examples of the pro region of the protein precursor facilitating the folding of the precursor as an intramolecular chaperone, of which pre-bovine pancreatic trypsin inhibitor is a well characterized one (61). Another role of the flexible C-peptide is probably related to allowing sufficient flexibility in the positioning of the chains for their optimal interaction. Thus, the constraints imposed by the short C-peptide in PIP, especially on the N-terminal region of the A chain, may disrupt the correct intra- and interchain interactions that sort out this region after the A20-B19 disulfide bridge forms. The fact that IGF-I tends to have folding isomers (52) lends support to the view that a shortened C-peptide constrains the folding process within the N-terminal A domain. There, the C-peptide of HPI seems to also act as a flexible tether and thus enhance folding by facilitating the molecular interaction between the A- and B-chain of HPI.

Although the C-peptide has no effects on the thermodynamic folding of PIP, it has profound effects on the kinetic folding pathway. How does the C-peptide affect the folding pathway of HPI? The proline isomerization is always a rate-limiting step in the folding reaction of small, single-domain proteins containing proline residue(s) (62). PIP has one proline residue at position B28, whereas HPI has three proline residues (the other two in the C-peptide), so the conformation formation of PIP is probably faster than HPI. During the refolding of PIP, the formation of native-like conformation may be faster than HPI. The native-like conformation will consequently guide the correct and hierarchical pairing of disulfide bonds. Thus, the different folding mechanism of HPI and PIP may be partially due to the proline isomerization.

Therefore, although the A- and B-chain of insulin contain sufficient information for the correct pairing of disulfide bonds (63, 64), we consider that the intact folding information of HPI resides in the A- and B-chain of insulin as well as the C-peptide.

Implications for the in Vivo Folding of HPI-- Only scrambled isomers in the refolding of HPI have been captured as intermediates, indicating that the free thiols on frdHPI prefer to form nonnative disulfide bonds rather than stand alone during the refolding process of HPI. The thiol-free scrambled disulfide isomer could preclude the intermolecular disulfide linkages for the off-pathway intermediates. In addition, the reshuffling of disulfide bonds in the disulfide isomers is an intramolecular reaction with high efficiency. Therefore, it is reasonable that the scrambled disulfide isomers could serve as folding intermediates in many other proteins such as hirudin, TAP, PCI, and epidermal growth factor (39, 65-67).

The foldability of proinsulin is limited in vitro in the absence of chaperones by aberrant aggregation of partially folded intermediates. Although the present studies were conducted at pH 10.0 to avoid such aggregation, the results have implications for folding under physiological conditions. HPI is synthesized in vivo as a preproinsulin with a signal peptide (16). The signal peptide then targets the pre-HPI for translocation into the endoplasmic reticulum, where the signal peptide region was cut off. Considering that glutathione is the principal redox buffer in endoplasmic reticulum (68) (similar to the redox condition we used here) and our results, we might imagine the refolding of HPI in vivo may adopt the following mechanism. During translation of the nascent peptide of HPI from ribosome into the endoplasmic reticulum, the six cysteines will be exposed to the endoplasmic reticulum one after another, and hence the CysB7 and CysB19 of HPI will be first exposed. Since the successive exposure of cysteines in A-chain must wait for the translocation of a 35-amino acid connecting peptide, the free CysB7 and CysB19 have the tendency to pair and form the disulfide bond B7-B19. The consecutively exposed cysteines on the A-chain will pair each other to form three consecutive disulfides like the intermediate P4. Thereafter, a series of scrambled disulfide isomers were formed like intermediate P1, P2, and P3 by disulfide bonds reshuffling. Along with forming secondary structure, the scrambled disulfide isomers, through reshuffling into native disulfide bonds, finally complete the folding. Despite the higher efficiency of HPI under the optimized conditions in in vitro experiments, the overall folding rate of HPI in these studies is much slower compared with the rate of protein synthesis and folding in vivo (69). This suggests that the folding of HPI in vivo requires the involvement of some cellular factors like protein-disulfide isomerase and chaperone. A recent study has shown that the catalytic activity and chaperone function of human protein-disulfide isomerase are required for the efficient refolding of proinsulin (70).

    ACKNOWLEDGEMENTS

We thank two anonymous reviewers for helpful advice and suggestions and Emily K. Collins for assistance with figures.

    FOOTNOTES

* This work was supported by National Foundation of Natural Science Grant 39670179 and Chinese Academy of Sciences Grant KJ951-B1-606.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 86-21-54921133; Fax: 86-21-64338357; E-mail: fengym@sunm.shcnc.ac.cn.

Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M300906200

    ABBREVIATIONS

The abbreviations used are: HPI, human proinsulin; PIP, porcine insulin precursor; IGF, insulin-like growth factor; IAA, sodium salt of idoacetic acid; frdHPI, fully reduced/denatured HPI; HPLC, high performance liquid chromatography; RP-HPLC, reverse phase HPLC; SS, disulfide bond; ESI-MS, electrospray ionization-mass spectrometry; HI, human insulin; DKP insulin, [AspB10, LysB28, ProB29] insulin mutant.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Steiner, D. F., and Chan, S. J. (1988) Horm. Metab Res. 20, 443-444[Medline] [Order article via Infotrieve]
2. Baker, E. N., Blundell, T. L., Cutfield, J. F., Cutfield, S. M., Dodson, E. J., Dodson, G. G., Hodgkin, D. M., Hubbard, R. E., Isaacs, N. W., and Reynolds, C. D. (1988) Philos. Trans. R. Soc. Lond. B Biol. Sci. 319, 369-456[Medline] [Order article via Infotrieve]
3. The Peking Insulin Structure Research Group. (1974) Sci. Sin. 17, 752-778
4. Roy, M., Lee, R. W., Brange, J., and Dunn, M. F. (1990) J. Biol. Chem. 265, 5448-5452[Abstract/Free Full Text]
5. Weiss, M. A., Hua, Q. X., Lynch, C. S., Frank, B. H., and Shoelson, S. E. (1991) Biochemistry 30, 7373-7389[Medline] [Order article via Infotrieve]
6. Olsen, H. B., Ludvigsen, S., and Kaarsholm, N. C. (1996) Biochemistry 35, 8836-8845[CrossRef][Medline] [Order article via Infotrieve]
7. Hua, Q. X., Nakagawa, S. H., Jia, W., Hu, S. Q., Chu, Y. C., Katsoyannis, P. G., and Weiss, M. A. (2001) Biochemistry 40, 12299-12311[CrossRef][Medline] [Order article via Infotrieve]
8. Hua, Q. X., Gozani, S. N., Chance, R. E., Hoffmann, J. A., Frank, B. H., and Weiss, M. A. (1995) Nat. Struct. Biol. 2, 129-138[Medline] [Order article via Infotrieve]
9. Hua, Q. X., Jia, W., Frank, B. H., Phillips, N. F., and Weiss, M. A. (2002) Biochemistry 41, 14700-14715[CrossRef][Medline] [Order article via Infotrieve]
10. Weiss, M. A., Hua, Q. X., Jia, W., Chu, Y. C., Wang, R. Y., and Katsoyannis, P. G. (2000) Biochemistry 39, 15429-15440[CrossRef][Medline] [Order article via Infotrieve]
11. Hua, Q. X., Narhi, L., Jia, W., Arakawa, T., Rosenfeld, R., Hawkins, N., Miller, J. A., and Weiss, M. A. (1996) J. Mol. Biol. 259, 297-313[CrossRef][Medline] [Order article via Infotrieve]
12. Hua, Q. X., Hu, S. Q., Frank, B. H., Jia, W., Chu, Y. C., Wang, S. H., Burke, G. T., Katsoyannis, P. G., and Weiss, M. A. (1996) J. Mol. Biol. 264, 390-403[CrossRef][Medline] [Order article via Infotrieve]
13. Guo, Z. Y., and Feng, Y. M. (2001) Biol. Chem. 382, 443-448[Medline] [Order article via Infotrieve]
14. Hua, Q. X., Chu, Y. C., Jia, W., Phillips, N. F., Wang, R. Y., Katsoyannis, P. G., and Weiss, M. A. (2002) J. Biol. Chem. 277, 43443-43453[Abstract/Free Full Text]
15. Steiner, D. F. (1967) Trans. N. Y. Acad. Sci. 30, 60-68[Medline] [Order article via Infotrieve]
16. Steiner, D. F., Cunningham, D., Spigelman, L., and Aten, B. (1967) Science 157, 697-700[Medline] [Order article via Infotrieve]
17. Steiner, D. F., Cho, S., Oyer, P. E., Terris, S., Peterson, J. D., and Rubenstein, A. H. (1971) J. Biol. Chem. 246, 1365-1374[Abstract/Free Full Text]
18. Steiner, D. F. (1978) Diabetes 27 Suppl. 1, 145-148[Medline] [Order article via Infotrieve]
19. Davidson, H. W., and Hutton, J. C. (1987) Biochem. J. 245, 575-582[Medline] [Order article via Infotrieve]
20. Davidson, H. W., Rhodes, C. J., and Hutton, J. C. (1988) Nature 333, 93-96[CrossRef][Medline] [Order article via Infotrieve]
21. Low, B. W., Fullerton, W. W., and Rosen, L. S. (1974) Nature 248, 339-340[Medline] [Order article via Infotrieve]
22. Steiner, D. F. (1973) Nature 243, 528-530[Medline] [Order article via Infotrieve]
23. Snell, C. R., and Smyth, D. G. (1975) J. Biol. Chem. 250, 6291-6295[Abstract]
24. Weiss, M. A., Frank, B. H., Khait, I., Pekar, A., Heiney, R., Shoelson, S. E., and Neuringer, L. J. (1990) Biochemistry 29, 8389-8401[Medline] [Order article via Infotrieve]
25. Anfinsen, C. B. (1973) Science 181, 223-230[Medline] [Order article via Infotrieve]
26. Matthews, C. R. (1993) Annu. Rev. Biochem. 62, 653-683[CrossRef][Medline] [Order article via Infotrieve]
27. Kim, P. S., and Baldwin, R. L. (1990) Annu. Rev. Biochem. 59, 631-660[CrossRef][Medline] [Order article via Infotrieve]
28. Creighton, T. E. (1986) Methods Enzymol. 131, 83-106[Medline] [Order article via Infotrieve]
29. Creighton, T. E., and Goldenberg, D. P. (1984) J. Mol. Biol. 179, 497-526[Medline] [Order article via Infotrieve]
30. Creighton, T. E. (1997) Biol. Chem. 378, 731-744[Medline] [Order article via Infotrieve]
31. Weissman, J. S., and Kim, P. S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9900-9904[Abstract]
32. Darby, N. J., Morin, P. E., Talbo, G., and Creighton, T. E. (1995) J. Mol. Biol. 249, 463-477[CrossRef][Medline] [Order article via Infotrieve]
33. Bulaj, G., and Goldenberg, D. P. (1999) Protein Sci. 8, 1825-1842[Abstract]
34. Weissman, J. S., and Kim, P. S. (1991) Science 253, 1386-1393[Medline] [Order article via Infotrieve]
35. Creighton, T. E., Darby, N. J., and Kemmink, J. (1996) FASEB J. 10, 110-118[Abstract/Free Full Text]
36. Xu, X., Rothwarf, D. M., and Scheraga, H. A. (1996) Biochemistry 35, 6406-6417[CrossRef][Medline] [Order article via Infotrieve]
37. Wedemeyer, W. J., Welker, E., Narayan, M., and Scheraga, H. A. (2000) Biochemistry 39, 4207-4216[CrossRef][Medline] [Order article via Infotrieve]
38. Rothwarf, D. M., and Scheraga, H. A. (1993) Biochemistry 32, 2671-2679[Medline] [Order article via Infotrieve]
39. Chang, J. Y., Schindler, P., Ramseier, U., and Lai, P. H. (1995) J. Biol. Chem. 270, 9207-9216[Abstract/Free Full Text]
40. Wu, J., Yang, Y., and Watson, J. T. (1998) Protein Sci. 7, 1017-1028[Abstract/Free Full Text]
41. Rinderknecht, E., and Humbel, R. E. (1978) J. Biol. Chem. 253, 2769-2776[Abstract]
42. Rinderknecht, E., and Humbel, R. E. (1978) FEBS Lett. 89, 283-286[CrossRef][Medline] [Order article via Infotrieve]
43. Bedarkar, S., Turnell, W. G., Blundell, T. L., and Schwabe, C. (1977) Nature 270, 449-451[Medline] [Order article via Infotrieve]
44. Chan, S. J., Cao, Q. P., and Steiner, D. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9319-9323[Abstract]
45. Nagasawa, H., Guo, F., Zhong, X. C., Xia, B. Y., Wang, Z. S., Qui, X. J., Wei, D. Y., Chen, E. I., Wang, J. Z., Suzuki, A., Isogai, A., Hori, Y., Tamura, S., and Ishizaki, H. (1980) Sci. Sin. 23, 1053-1060[Medline] [Order article via Infotrieve]
46. Smit, A. B., Vreugdenhil, E., Ebberink, R. H., Geraerts, W. P., Klootwijk, J., and Joosse, J. (1988) Nature 331, 535-538[CrossRef][Medline] [Order article via Infotrieve]
47. Bullesbach, E. E., Schwabe, C., and Lacy, E. R. (1997) Biochemistry 36, 10735-10741[CrossRef][Medline] [Order article via Infotrieve]
48. Milner, S. J., Carver, J. A., Ballard, F. J., and Francis, G. L. (1999) Biotechnol. Bioeng. 62, 693-703[CrossRef][Medline] [Order article via Infotrieve]
49. Miller, J. A., Narhi, L. O., Hua, Q. X., Rosenfeld, R., Arakawa, T., Rohde, M., Prestrelski, S., Lauren, S., Stoney, K. S., Tsai, L., and Weiss, M. A. (1993) Biochemistry 32, 5203-5213[Medline] [Order article via Infotrieve]
50. Yang, Y., Wu, J., and Watson, J. T. (1999) J. Biol. Chem. 274, 37598-37604[Abstract/Free Full Text]
51. Rosenfeld, R. D., Miller, J. A., Narhi, L. O., Hawkins, N., Katta, V., Lauren, S., Weiss, M. A., and Arakawa, T. (1997) Arch. Biochem. Biophys. 342, 298-305[CrossRef][Medline] [Order article via Infotrieve]
52. Hober, S., Forsberg, G., Palm, G., Hartmanis, M., and Nilsson, B. (1992) Biochemistry 31, 1749-1756[Medline] [Order article via Infotrieve]
53. Guo, Z. Y., Shen, L., and Feng, Y. M. (2002) Biochemistry 41, 1556-1567[CrossRef][Medline] [Order article via Infotrieve]
54. Steiner, D. F., and Clark, J. L. (1968) Proc. Natl. Acad. Sci. U. S. A. 60, 622-629[Medline] [Order article via Infotrieve]
55. Qiao, Z. S., Guo, Z. Y., and Feng, Y. M. (2001) Biochemistry 40, 2662-2668[CrossRef][Medline] [Order article via Infotrieve]
56. Frank, B. H., Veros, A. J., and Pekar, A. H. (1972) Biochemistry 11, 4926-4931[Medline] [Order article via Infotrieve]
57. Baldwin, R. L. (1989) Trends Biochem. Sci. 14, 291-294[CrossRef][Medline] [Order article via Infotrieve]
58. Baker, D. (2000) Nature 405, 39-42[CrossRef][Medline] [Order article via Infotrieve]
59. Alm, E., and Baker, D. (1999) Curr. Opin. Struct. Biol. 9, 189-196[CrossRef][Medline] [Order article via Infotrieve]
60. Chen, L. M., Yang, X. W., and Tang, J. G. (2002) J. Biochem. (Tokyo) 131, 855-859[Abstract]
61. Weissman, J. S., and Kim, P. S. (1992) Cell 71, 841-851[Medline] [Order article via Infotrieve]
62. Kim, P. S., and Baldwin, R. L. (1982) Annu. Rev. Biochem. 51, 459-489[CrossRef][Medline] [Order article via Infotrieve]
63. Wang, C. C., and Tsou, C. L. (1991) Trends Biochem. Sci. 16, 279-281[CrossRef][Medline] [Order article via Infotrieve]
64. Tang, J. G., and Tsou, C. L. (1990) Biochem. J. 268, 429-435[Medline] [Order article via Infotrieve]
65. Chang, J. Y. (1996) Biochemistry 35, 11702-11709[CrossRef][Medline] [Order article via Infotrieve]
66. Chatrenet, B., and Chang, J. Y. (1993) J. Biol. Chem. 268, 20988-20996[Abstract/Free Full Text]
67. Chang, J. Y., Canals, F., Schindler, P., Querol, E., and Aviles, F. X. (1994) J. Biol. Chem. 269, 22087-22094[Abstract/Free Full Text]
68. Hwang, C., Sinskey, A. J., and Lodish, H. F. (1992) Science 257, 1496-1502[Medline] [Order article via Infotrieve]
69. Gething, M. J., and Sambrook, J. (1992) Nature 355, 33-45[CrossRef][Medline] [Order article via Infotrieve]
70. Winter, J., Klappa, P., Freedman, R. B., Lilie, H., and Rudolph, R. (2002) J. Biol. Chem. 277, 310-317[Abstract/Free Full Text]
71. Yan, H., Guo, Z. Y., Gong, X. W., Xi, D., and Feng, Y. M. (2003) Protein Sci. 12, 768-775[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.