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• W2016643526 abstract "Human epidermal growth factor (EGF) contains three disulfides and 53 amino acids. Reduced/denatured EGF refolds spontaneously in vitro to acquire its native structure. The mechanism of this folding process has been elucidated by structural analysis of both acid and iodoacetate trapped intermediates. The results reveal that the folding is accompanied by a sequential flow of unfolded EGF (0-disulfide) through three groups of folding intermediates, namely 1-disulfide, 2-disulfide, and 3-disulfide (scrambled) EGF isomers, to reach the native structure. Equilibrium occurs among isomers of each class of disulfide species, and the composition of intermediates appears to be highly heterogeneous. Together, at least 27 fractions of folding intermediates have been identified, but there exist only limited numbers of well populated species which constitute more than 80% of the total intermediates found during EGF folding.Six species of such well populated intermediates have been isolated, which included two 1-S-S, two 2-S-S, and two 3-S-S scrambled species. Their disulfide structures have been identified here. Both 1-S-S isomers are found to contain non-native disulfides. One of the 2-S-S species consists of two non-native disulfides and the other admits two native disulfides. Among the six disulfides of the two scrambled species, only one is native. Together, native disulfides constitute 25% of the total disulfides found in these six well populated intermediates. These results contrast sharply to those observed with bovine pancreatic trypsin inhibitor, which has shown that well populated folding intermediates consist of exclusively native disulfides (Weissman, J. S., and Kim, P. S. (1991) Science 253, 1386-1393). We propose that well populated folding intermediates, regardless of whether they contain native or non-native disulfides, do not necessarily represent the productive species and specify the folding pathway.Furthermore, conditions influencing the efficiency of EGF folding have been investigated. It is demonstrated here that under optimized compositions of redox agents, including the use of cysteine/cystine and protein disulfide isomerase, the in vitro folding of EGF could be achieved quantitatively within 1 min. Human epidermal growth factor (EGF) contains three disulfides and 53 amino acids. Reduced/denatured EGF refolds spontaneously in vitro to acquire its native structure. The mechanism of this folding process has been elucidated by structural analysis of both acid and iodoacetate trapped intermediates. The results reveal that the folding is accompanied by a sequential flow of unfolded EGF (0-disulfide) through three groups of folding intermediates, namely 1-disulfide, 2-disulfide, and 3-disulfide (scrambled) EGF isomers, to reach the native structure. Equilibrium occurs among isomers of each class of disulfide species, and the composition of intermediates appears to be highly heterogeneous. Together, at least 27 fractions of folding intermediates have been identified, but there exist only limited numbers of well populated species which constitute more than 80% of the total intermediates found during EGF folding. Six species of such well populated intermediates have been isolated, which included two 1-S-S, two 2-S-S, and two 3-S-S scrambled species. Their disulfide structures have been identified here. Both 1-S-S isomers are found to contain non-native disulfides. One of the 2-S-S species consists of two non-native disulfides and the other admits two native disulfides. Among the six disulfides of the two scrambled species, only one is native. Together, native disulfides constitute 25% of the total disulfides found in these six well populated intermediates. These results contrast sharply to those observed with bovine pancreatic trypsin inhibitor, which has shown that well populated folding intermediates consist of exclusively native disulfides (Weissman, J. S., and Kim, P. S. (1991) Science 253, 1386-1393). We propose that well populated folding intermediates, regardless of whether they contain native or non-native disulfides, do not necessarily represent the productive species and specify the folding pathway. Furthermore, conditions influencing the efficiency of EGF folding have been investigated. It is demonstrated here that under optimized compositions of redox agents, including the use of cysteine/cystine and protein disulfide isomerase, the in vitro folding of EGF could be achieved quantitatively within 1 min. Intermediates that occur in the folding pathway of disulfide-containing proteins were recognized in the pioneering work on bovine pancreatic trypsin inhibitor (BPTI) 1The abbreviations used are: BPTIbovine pancreatic trypsin inhibitorEGFrecombinant human epidermal growth factorGSHreduced glutathioneGSSGoxidized glutathioneCyscysteineCys-CyscystineMALDImatrix-assisted laser desorption ionizationGdmClguanidinium chlorideHPLChigh performance liquid chromatographyPTHphenylthiohydantoin. (Creighton, 1978Creighton T.E. Prog. Biophys. Mol. Biol. 1978; 33: 231-297Google Scholar, Creighton, 1990Creighton T.E. Biochem. J. 1990; 270: 1-16Google Scholar) and ribonuclease A (Creighton, 1979Creighton T.E. J. Mol. Biol. 1979; 129: 411-431Google Scholar; Konishi et al., 1981Konishi Y. Ooi T. Scheraga H.A. Biochemistry. 1981; 20: 3945-3955Google Scholar; Scheraga et al., 1984Scheraga H.A. Konishi Y. Ooi T. Adv. Biophys. 1984; 18: 21-41Google Scholar). In the case of BPTI, eight (including native) disulfide-bonded intermediates out of 75 possible species were initially described (Creighton, 1978Creighton T.E. Prog. Biophys. Mol. Biol. 1978; 33: 231-297Google Scholar; Creighton and Goldenberg, 1984Creighton T.E. Goldenberg D.P. J. Mol. Biol. 1984; 179: 497-524Google Scholar). Some of those well populated 1- and 2-disulfide intermediates appeared to contain non-native disulfides and were proposed to be involved in the process of folding. This original BPTI model was recently re-examined using modern separation and analytical methodologies. In that study (Weissman and Kim, 1991Weissman J.S. Kim P.S. Science. 1991; 253: 1386-1393Google Scholar), it was concluded that all well populated folding intermediates consisted of only native disulfide bonds. Raging debates ensued as a consequence of these discrepancies (Creighton, 1992Creighton T.E. Science. 1992; 256: 111-112Google Scholar; Weissman and Kim, 1992Weissman J.S. Kim P.S. Science. 1992; 256: 112-114Google Scholar), and discussions are focused mainly upon the importance of intermediates containing non-native disulfides. In those studies, however, no non-native 3-disulfide intermediates have been described. bovine pancreatic trypsin inhibitor recombinant human epidermal growth factor reduced glutathione oxidized glutathione cysteine cystine matrix-assisted laser desorption ionization guanidinium chloride high performance liquid chromatography phenylthiohydantoin. On the contrary, kinetically trapped non-native 3-disulfide (scrambled) intermediates were detected and characterized in recent studies of recombinant hirudin (Chatrenet and Chang, 1992Chatrenet B. Chang J.-Y. J. Biol. Chem. 1992; 267: 3038-3043Google Scholar, Chatrenet and Chang, 1993Chatrenet B. Chang J.-Y. J. Biol. Chem. 1993; 268: 20988-20996Google Scholar) and potato carboxypeptidase inhibitor (Chang et al., 1994Chang J.-Y. Canals F. Schindler P. Querol E. Aviles F.X. J. Biol. Chem. 1994; 269: 22087-22094Google Scholar). They were found reproducibly in high concentrations and were observed under a wide range of folding conditions, including those favorable conditions which permit regeneration of the native protein to be completed within 30 s. Furthermore, the level of accumulation of the scrambled species has been shown to depend upon the redox potential applied and thus could be experimentally manipulated (Chang, 1994Chang J.-Y. Biochem. J. 1994; 300: 643-650Google Scholar). In one case, more than 98% of the total sample was found to be trapped as scrambled intermediates before trace amount of the native structure even appeared (Chang et al., 1994Chang J.-Y. Canals F. Schindler P. Querol E. Aviles F.X. J. Biol. Chem. 1994; 269: 22087-22094Google Scholar). These findings indicate that scrambled proteins may play an essential role along the pathway of productive folding. This proposal, however, contradicts conventional wisdom which considers scrambled species as abortive structures of “off-pathway” folding. One may further suggest that the presence of scrambled intermediates is not a general phenomenon and represents only isolated, unusual cases for hirudin and potato carboxypeptidase inhibitor. In order to clarify these uncertainties, studies on disulfide folding pathway using other comparable proteins are required. In this report, we use recombinant human epidermal growth factor (EGF), which is also a small, compact protein (53 amino acid residues), and like hirudin, contains only antiparallel β-sheet and no α-helix (Carver et al., 1986Carver J.A. Cooke R.M. Esposito G. Campbell I.D. Gregory H. Sheard B. FEBS Lett. 1986; 205: 77-81Google Scholar; Montelione et al., 1987Montelione G.T. Wuethrich K. Nice E.C. Burgess A.W. Scheraga H.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5226-5230Google Scholar), as an example to study the behavior of folding intermediates and confirm the formation of non-native 3-disulfide in-termediates during folding. We also examine conditions that enhance the efficiency of EGF folding. Recombinant human epidermal growth factor (EGF) was derived from Escherichia coli. Cells and was supplied by Protein Institute Inc., Broomall, PA. The purity was greater than 98% as judged by SDS-polyacrylamide gel electrophoresis and N-terminal sequence analysis. The recombinant EGF is fully biological active when compared with standards. Reduced glutathione (GSH), oxidized glutathione (GSSG), cysteine (Cys), cystine (Cys-Cys), thermolysin (P-1512), and Glu-C protease were obtained from Sigma. Protein disulfide isomerase (number 7318) was purchased from Takara, Kyoto, Japan. Control foldings are those performed either in Tris-HCl buffer alone (control -) or in the same buffer containing 0.25 m M of 2-mercaptoethanol (control +). Results of control foldings serve as standards for measuring the efficiencies of EGF folding in the presence of various redox agents. EGF (1.5 mg) was dissolved in 0.5 ml of Tris-HCl buffer (0.5 M, pH 8.5) containing 5 M of GdmCl and 30 m M of dithiothreitol. Reduction and denaturation of EGF was carried out at 22°C for 90 min. To initiate the folding, the sample was passed through a PD-10 column (Pharmacia) equilibrated in 0.1 M Tris-HCl buffer, pH 8.5. Desalting took about 1 min and unfolded EGF was recovered in 1.1 ml, which was immediately diluted with the same Tris-HCl buffer to a final protein concentration of 1 mg/ml, both in the absence (control -) and presence (control +) of 0.25 m M 2-mercaptoethanol. Folding intermediates were trapped in a time course manner by mixing aliquots of the sample with an equal volume of (a) 4% trifluoroacetic acid in water (reversible trapping) or (b) 0.4 M iodoacetic acid in the Tris-HCl buffer (0.5 M, pH 8.5) (irreversible trapping). In the case of iodoacetate trapping, carboxymethylation was performed at 22°C for 30 min, followed by desalting using the PD-10 column. Trapped folding intermediates were separated by HPLC. The procedures of unfolding and refolding are as those described in the control folding experiments. Selected concentrations of redox agents or denaturants were introduced immediately after unfolded EGF was desalted through the PD-10 column. Folding intermediates were trapped reversibly or irreversibly as those described above. Six fractions of well populated intermediates (I-A, I-B, II-A, II-B, III-A, and III-B), derived from the “control -” folding and trapped by iodoacetate, were isolated for structural analysis. 1-Disulfide intermediates (I-A and I-B) (3 μg) were digested with 0.3 μg of Glu-C protease or trypsin in 30 μl of ammonium bicarbonate solution (50 m M, pH 8.0) for 16 h at 23°C. In this case, fully reduced carboxymethylated EGF was processed in parallel as a control. The samples were then acidified with an equal volume of 4% trifluoroacetic acid and directly subjected to automatic sequencing. 2- and 3-disulfide intermediates (~30 μg) were treated with 3 μg of thermolysin in 100 μl of N-ethylmorpholine/acetate buffer (50 m M, pH 6.4). Digestion was carried out at 23°C for 16 h. Peptides were then isolated by HPLC and analyzed by amino acid sequencing and mass spectrometry. Amino analysis was performed with the dabsyl chloride precolumn derivatization method (Chang and Knecht, 1991Chang J.-Y. Knecht R. Anal. Biochem. 1991; 197: 52-58Google Scholar), which permits direct evaluation of the disulfide (cystine) content. Amino acid sequencing was done with either an Applied Biosystems 470A sequencer or a Hewlett-Packard G-1000A sequencer. The digests of I-A and I-B were mainly analyzed by the HP sequencer, because it gives more reliable quantitation on the recovery of PTH-Cys(Cm). Cystine-containing peptides were mostly analyzed by the ABI instrument. An internal standard, 2-nitroacetophenone, which eluted in between PTH-His and PTH-Tyr was introduced in order to ensure precise quantitation of PTH derivatives (Ramseier and Chang, 1994Ramseier U. Chang J.-Y. Anal. Biochem. 1994; 221: 231-233Google Scholar). It was predissolved in the solvent (2 μM) which transfers PTH derivatives from the conversion flask to the HPLC. During the analysis of cystine containing peptides, a unique signal di-PTH-cystine appeared when both half-cystines were recovered in the same degradation cycle (Haniu et al., 1994Haniu M. Acklin C. Kenney W. Rohde M.F. Int. J. Peptide Protein Res. 1994; 43: 81-86Google Scholar). di-PTH-cystine is eluted near PTH-Tyr, but can be easily distinguished from the tyrosine derivative by an additional absorbance at 313 nm. The MALDI mass spectrometer was a home-built time of flight instrument with a nitrogen laser of 337-nm wavelength and 3-ns pulse width. The apparatus has been described in detail elsewhere (Boernsen et al., 1990Boernsen K.O. Schaer M. Widmer M. Chimia. 1990; 44: 412-416Google Scholar). The calibration was performed either externally or internally, by using standard proteins (hypertensin, Mr1031.19; synacthen, Mr2934.50 and calcitonin, Mr3418.91). Analysis of the iodoacetate-trapped folding intermediates is further explained in the legend of Fig. 1. The biological activity of recombinant EGF was compared with the standard recombinant EGF using the assay method described (Savage et al., 1973Savage Jr., C.R. Hash J.H. Cohen S. J. Biol. Chem. 1973; 248: 7669-7672Google Scholar). The ED50as determined by the dose-dependent stimulation of thymidine uptake by Balb/c 3T3 cells is 2.0 ng/ml. Refolded EGF was compared with a standard sample by an HPLC stability-indicating assay. The fully biological active EGF samples and standard were assayed by reversed phase HPLC described in the legend of Fig. 2. Refolded native EGF is assessed by comparing their HPLC with that of the standard. Folding intermediates of EGF were first analyzed for their disulfide contents in order to evaluate the rate of disulfide formation during the folding. This was done with amino acid composition analysis (Chang and Knecht, 1991Chang J.-Y. Knecht R. Anal. Biochem. 1991; 197: 52-58Google Scholar). Two sets of samples obtained from the folding experiments performed in the absence (control -) and presence (control +) of 2-mercaptoethanol (0.25 m M) were analyzed. The results showed that: (a) the decrease of cysteine (detected in the form of carboxymethylcysteine) was quantitatively accounted for by the recovery of disulfide, and (b) the rate of total disulfide recovery remained indistinguishable regardless of whether the folding was carried out in the absence or presence of 2-mercaptoethanol. In both experiments, three intact disulfides formed after 24 h of folding (data not shown). The data for Cys/Cys-Cys composition played a crucial role in the identification of scrambled EGF. It was subsequently revealed by HPLC analysis that the yield of native EGF was indeed dependent upon the presence of 2-mercaptoethanol. In the presence of 2-mercaptoethanol, or Cys, or reduced glutathione, the formation of three disulfide bonds was accompanied by the quantitative recovery of native EGF. Without 2-mercapto-ethanol, about 45% of the 3-disulfide EGF were trapped as species distinguishable from the native one (see Fig. 2, 24-h samples). These trapped EGF species are scrambled non-native 3-disulfide species. Folding intermediates of EGF were further characterized by MALDI mass spectrometry in order to determine the concentrations of disulfide species presented in the intermediates. The results were obtained from samples folded in the buffer alone (control -) and trapped by iodoacetic acid. The data (Fig. 1) demonstrate a sequential flow of unfolded EGF through 1- and 2-disulfide intermediates to the 3-disulfide species. The high level of accumulation of 2-disulfide intermediates indicated that the conversion of 2-disulfide species to 3-disulfide species constituted one of the major rate-limiting steps of EGF folding. The 24-h folded sample was shown to contain virtually only 3-disulfide species which further confirmed that the non-native species trapped in the 24-h sample (Fig. 2) are the scrambled EGF. Folding intermediates of EGF were analyzed by HPLC. The raw data, presented in Fig. 2, were obtained from the samples of control - experiment trapped either by acid (right column) or by iodoacetic acid (left column). In order to be able to interpret these chromatograms, structural information of the fractionated intermediates was required. Therefore, 18 fractions of intermediates were first isolated from the 30-min iodoacetate-trapped sample (Fig. 3) and analyzed by mass spectrometry. Concentrations of disulfide species presented in each of those fractions are given in Fig. 4. The results revealed that this sample comprised a minimum of seven 1-disulfide isomers and 13 2-disulfide isomers. Most 1-disulfide species eluted at fractions 16 (I-A) and 17 (I-B) and 2-disulfide species mostly accumulated within fractions 3 (II-A) and 6 (II-B). Similar analysis of the 7-h and 48-h samples (Fig. 3) showed that fractions 4 (III-A) and 5 (III-B) contained predominantly (>92%) 3-disulfide scrambled species. The three groups of intermediates were extensively overlapped, but predominant fractions of these three disulfide species were fortunately well separated. It was also apparent that along the folding process, equilibrium existed among isomers of each disulfide species. For instance, the concentration of 2-disulfide intermediates ascended and then descended as folding progressed, but the relative ratio of fractions 3, 6, and 7 (which contained exclusively 2-disulfide species) remained constant. Scrambled 3-disulfide species and 1-disulfide species behaved similarly during the folding.Figure 4:Analysis by mass spectrometry of folding intermediates of EGF isolated by HPLC. Eighteen fractions were isolated from the 30-min sample (trapped by iodoacetate) (see Fig. 3) and analyzed by MALDI mass spectrometry in order to determine the disulfide species contained in each fraction. The content of disulfide species is determined by the mass peak height and expressed as percentage in each fraction. The data should be allowed a standard deviation of ± 10%. Fractions 4 and 5 contain only minute amounts of intermediates and are shown to be comprised of about 50% each of 2-disulfide and 3-disulfide (scrambled) species. A separate analysis of the 7-h trapped sample shows that fractions 4 and 5 contain predominantly (>90%) 3-disulfide species.View Large Image Figure ViewerDownload (PPT) These data demonstrated that the folding pathway of EGF was characterized by a sequential flow of unfolded EGF (R) through three groups of equilibrated intermediates, namely, 1-disulfide, 2-disulfide, and 3-disulfide (scrambled) isomers. With the control - folding experiment, 45% of the folding intermediates were stuck as scrambled species (see Fig. 2, 24-h sample), unable to convert to the native EGF due to the lack of free thiols to catalyze their disulfide reshuffling. This problem was overcome by including 2-mercaptoethanol (data not shown) in the folding buffer, in which recoveries of native EGF were found to be greater than 96% after 24 h of folding. The HPLC profiles of acid-trapped intermediates (Fig. 2, right column) did not fully resemble those of iodoacetate-trapped counterparts. Notably, most acid-trapped 2-disulfide species were eluted under the same fraction (the peak marked as II). Thus, interpretation of EGF folding based on the analysis of acid-trapped samples can be very tricky. The predominance of a single peak containing 2-disulfide species can easily mislead to the simplification that folding of EGF undergoes only one species of 2-disulfide intermediates. The pattern of the 24-h acid-trapped sample is indistinguishable from that of iodoacetate-trapped sample, because both contained only 3-disulfide EGF. This can be achieved by a number of strategies. The most common one is “peptide mapping.” This requires isolation and analysis of every enzyme fragmented peptides, as will be shown in the following section. Alternatively, it can be done by selective labeling of disulfide bonds (after reduction) with a color (Chang, 1993Chang J.-Y. J. Biol. Chem. 1993; 268: 4043-4049Google Scholar) or fluorescent thiol-specific reagents (Weissman and Kim, 1991Weissman J.S. Kim P.S. Science. 1991; 253: 1386-1393Google Scholar). Both methods need microgram amounts of the intermediates, HPLC separation of peptides, and numerous attempts of sequence analysis. The most sensitive and effective method, however, is to take the advantage of modern Edman chemistry and the known sequence of EGF by direct sequencing of the peptide mixture of 1-disulfide intermediates. This strategy is sketched in Fig. 5 and described as follows: 1) select an enzyme that will produce a mixture of peptides, with all cysteines located at different positions in the peptide sequences; 2) subject the peptide mixture to automatic sequencing and quantitate recoveries of PTH-Cys(Cm) at expected cycles of Edman degradation. Cysteines which are not involved in disulfide pairings will be recovered as PTH-Cys(Cm), and those engaged in the disulfide linking will generate a blank gap; 3) compare the results obtained from the folding intermediates to that of control sample (fully reduced carboxymethylated EGF). This method requires low picomoles (nanograms) of samples, no HPLC separation of peptides and basically only one sequence analysis for each intermediate. For EGF, such peptide mixtures could be generated by either trypsin or Glu-C protease digestion (Fig. 5). The sequencing data obtained from the analysis of Glu-C digests are given in Fig. 6. It shows unambiguously that I-A and I-B contain Cys14-Cys24and Cys6-Cys14, respectively, both are non-native disulfides (Fig. 8). The results obtained from trypsin digests are equally conclusive (data not shown).Figure 8:Disulfide structures of six well populated folding intermediates of EGF. The arrows do not imply the direct conversion between the indicated species.View Large Image Figure ViewerDownload (PPT) The choice of methods for elucidating the disulfide structures of 2- and 3-disulfide intermediates is limited to the technique of peptide mapping. In this approach, selection of enzymes is critical. The digestion should be carried out at neutral or acidic pH and allow at least partial cleavage at peptide bonds between all neighboring cysteines. Thermolysin has been found to be an ideal enzyme for this purpose. Peptides were separated by HPLC (Fig. 7). Distinctions between cystine- and non-cystine-containing peptides can be generally recognized. Those which do not appear constantly in all mappings most likely contain disulfides. All peptides were analyzed by amino acid sequencing and mass spectrometry. Crucial data which permit assignments of disulfide pairings are presented in Table I. Two cystine peptides, with nearly equal recoveries and corresponding to two native disulfides, Cys14-Cys31and Cys33-Cys42, were found in II-A-7 and II-A-12, respectively. Despite the shoulder peak of II-A-12, sequence and mass analysis have revealed no contaminants of minor sequences. The two disulfide bonds of species II-B were also found in two major peaks. II-B-15 consisted of three peptides linked by two disulfide bridges, which could be oriented in a combination of either Cys14-Cys33/Cys24-Cys31or Cys14-Cys31/Cys24-Cys33. The finding of Cys14-Cys33in peak II-B-7 confirms that the former structure is the correct one. In this intermediate, both disulfides are non-native. Scrambled EGFs are 3-disulfide species. For III-A, the disulfides were detected in five peaks. Cys14-Cys33and Cys24-Cys31were identified in III-A-5 and III-A-10 (Table I). Cys6-Cys42was found in three different peaks (III-A-13, III-A-15, and III-A-17), due to nonspecific cleavages by thermolysin. In III-A, all disulfides are non-native. III-B is the most predominant scrambled species. Its three disulfides were recovered in four major peaks. Cys24-Cys31was found in III-B-2 and Cys33-Cys42was detected in III-B-8. The third disulfide of III-B, Cys6-Cys14, was found in III-B-14 as well as the tailing shoulder (right-hand) of III-B-11 (Table I). For all four well populated intermediates, there is no evidence of contamination of minor species (<10%). The results of their disulfide structures are summarized in Fig. 8.Table I:Structures of the disulfide containing peptides derived from the well populated folding intermediates of EGF Open table in a new tab Two systems of redox agents, GSH/GSSG and Cys/Cys-Cys were evaluated here. The effect of GSH was found to be similar to that of 2-mercaptoethanol which was to promote the conversion of scrambled EGF to the native EGF. In achieving this, it neither accelerated the flow of intermediates between unfolded species and scrambled species nor altered the patterns of intermediates compositions. The only obvious difference between those performed with and without GSH was the level of accumulation of scrambled species and the recovery of native EGF. Without GSH (control -), about 50% of EGF was trapped as scrambled species. In the presence of GSH, the yield of native EGF was nearly quantitative after 24 h of folding. GSSG played a different role. It enhanced the flow of intermediates between 0-disulfide EGF and scrambled species and as a consequence also accelerated the recovery of native EGF during the early phase of folding. In the presence of 0.5 m M of GSSG, the only detectable intermediates after 3 h of folding were scrambled species, and a substantial portion of scrambled EGF also become trapped, unable to convert to the native EGF even after 24 h of folding under these conditions. By including a mixture of GSH/GSSG in the folding solution, both the flow of intermediates and the conversion of scrambled EGF to the native EGF were accelerated. Under these conditions, folding of EGF was achieved quantitatively within 4 h. Cys/Cys-Cys also regulated the folding of EGF through a similar mechanism, except that it is more potent than the GSH/GSSG system. Direct comparison of the Cys-Cys and GSSG indicated that the former was about 5-10-fold more effective (at equal molar basis) in promoting the flow of intermediates to the 3-disulfide states (Fig. 9). In another experiment, it was demonstrated that trapped scrambled EGF species were able to reshuffle their mismatched disulfides to acquire the native structure within 1 h when 1 m M of Cys was introduced. Along this process of reorganization (consolidation), scrambled species remained in equilibrium. The above findings suggested that both the speed of EGF folding and the recovery of native EGF could be greatly improved under optimized compositions of redox agents. To demonstrate this potential, unfolded EGF was refolded in the Tris-HCl buffer containing 4 M sodium chloride and in the presence of the following redox systems: (a) GSH/GSSG (4 m M/2 m M); (b) Cys/Cys-Cys (4 m M/2 m M), and (c) Cys/Cys-Cys (4 m M/2 m M) plus protein disulfide isomerase (40 μM). Selection of these conditions was intended to (a) allow head-on comparison of the potencies between the GSH/GSSG and Cys/Cys-Cys systems and (b) assess the efficacy of protein disulfide isomerase (Epstein et al., 1963Epstein C.J. Goldberger R.F. Anfinsen C.B. Cold Spring Harbor Symp. Quant. Biol. 1963; 28: 439-449Google Scholar; Freedman, 1984Freedman R.B. Trends Biochem. Sci. 1984; 9: 438-441Google Scholar; Bulleid, 1993Bulleid N.J. Adv. Prot. Chem. 1993; 44: 125-150Google Scholar). The outcome was judged by the rate of the recovery of native EGF. It revealed that Cys/Cys-Cys was 10-fold more effective than GSH/GSSG in promoting the formation of native EGF. The improvement was multiplied by another 7-fold when 40 μM of protein disulfide isomerase was added. Under these optimized conditions, folding of EGF completed within one minute (Fig. 10). Denaturants (8 M urea or 5 M GdmCl) were included in the folding buffer in order to examine their effects on the folding mechanism of EGF. EGF was allowed to refold in the presence of 8 M urea without or with 0.25 m M of 2-mercaptoethanol (8 M urea - and 8 M urea +). These two experiments were repeated in the presence of 5 M GdmCl (5 M GdmCl - and 5 M GdmCl +). Folding intermediates were trapped by iodoacetic acid and analyzed by HPLC. T" @default.
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• W2016643526 title "The Disulfide Folding Pathway of Human Epidermal Growth Factor" @default.
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• W2016643526 cites W1501624911 @default.
• W2016643526 cites W1552905434 @default.
• W2016643526 cites W1651750842 @default.
• W2016643526 cites W1900519315 @default.
• W2016643526 cites W1983882926 @default.
• W2016643526 cites W1988397041 @default.
• W2016643526 cites W1991498388 @default.
• W2016643526 cites W1997500814 @default.
• W2016643526 cites W1999473806 @default.
• W2016643526 cites W2001451008 @default.
• W2016643526 cites W2009543203 @default.
• W2016643526 cites W2015561631 @default.
• W2016643526 cites W2019139743 @default.
• W2016643526 cites W2026274208 @default.
• W2016643526 cites W2040550807 @default.
• W2016643526 cites W2045777307 @default.
• W2016643526 cites W2073084525 @default.
• W2016643526 cites W2081871033 @default.
• W2016643526 cites W2081945921 @default.
• W2016643526 cites W2084652190 @default.
• W2016643526 cites W2093064306 @default.
• W2016643526 cites W2139327615 @default.
• W2016643526 cites W2326282683 @default.
• W2016643526 cites W2407474667 @default.
• W2016643526 cites W2951797907 @default.
• W2016643526 cites W3207390656 @default.
• W2016643526 cites W4213114154 @default.
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