In vitro insulin refolding: Characterization of the intermediates and the putative folding pathway

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates: two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient I was formed from P3A through thiol/disulfide exchange reaction; then, transients II and III, each containing two native disulfides, were formed through the recognition and interaction of transient I with P4B or P6B and the thiol group's oxidation reaction mainly using GSSG as oxidative reagent; finally, transients II and III, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.     

<Emphasis Type="Italic">In vitro</Emphasis> insulin refolding: Characterization of the intermediates and the putative folding pathway

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates: two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient I was formed from P3A through thiol/disulfide exchange reaction; then, transients II and III, each containing two native disulfides, were formed through the recognition and interaction of transient I with P4B or P6B and the thiol group’s oxidation reaction mainly using GSSG as oxidative reagent; finally, transients II and III, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.     

Unassisted refolding of urea-denatured arginine kinase from shrimp Feneropenaeus chinensis: evidence for two equilibrium intermediates in the refolding pathway

The refolding process and the equilibrium intermediates of urea-denatured arginine kinase (AK) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) intrinsic fluorescence, far-UV circular dichroism (CD), size-exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and N') were discovered, and a refolding scheme of urea-denatured AK was proposed. During the refolding of urea-denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (N') existed between a 0.4- and 0.8-M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (K(D)) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea-denatured AK was in two-phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea-denatured AK contained at the least two equilibrium refolding intermediates.     

In vitro refolding of human proinsulin. Kinetic intermediates,putative disulfide-forming pathway folding initiation site,and potential role of C-peptide in folding process

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.     

The disulfide-coupled folding pathway of apamin as derived from diselenide-quenched analogs and intermediates.

The sequence of apamin, an 18 residue bee venom toxin, encloses all the information required for the correct disulfide-coupled folding into the cystine-stabilized alpha-helical motif. Three apamin analogs, each containing a pair of selenocysteine residues replacing the related cysteines, were synthesized to mimic the three possible apamin isomers with two crossed, parallel, or consecutive disulfides, respectively. Refolding experiments clearly revealed that the redox potential of selenocysteine prevails over the sequence encoded structural information for proper folding of apamin. Thus, selenocysteine can be used as a new device to generate productive and nonproductive folding intermediates of peptides and proteins. In fact, disulfides are selectively reduced in presence of the diselenide and the conformational features derived from these intermediates as well as from the three-dimensional (3D) structures of the selenocysteine-containing analogs with their nonnatural networks of diselenide/disulfide bridges allowed to gain further insight into the subtle driving forces for the correct folding of apamin that mainly derive from local conformational preferences.     

Early intermediates in the PDI-assisted folding of ribonuclease A

The oxidative refolding of ribonuclease A has been investigated in several experimental conditions using a variety of redox systems. All these studies agree that the formation of disulfide bonds during the process occurs through a nonrandom mechanism with a preferential coupling of certain cysteine residues. We have previously demonstrated that in the presence of glutathione the refolding process occurs through the reiteration of two sequential reactions: a mixed disulfide with glutathione is produced first which evolves to form an intramolecular S-S bond. In the same experimental conditions, protein disulfide isomerase (PDI) was shown to catalyze formation and reduction of mixed disulfides with glutathione as well as formation of intramolecular S-S bonds. This paper reports the structural characterization of the one-disulfide intermediate population during the oxidative refolding of Ribonuclease A under the presence of PDI and glutathione with the aim of defining the role of the enzyme at the early stages of the reaction. The one-disulfide intermediate population occurring at the early stages of both the uncatalyzed and the PDI-catalyzed refolding was purified and structurally characterized by proteolytic digestion followed by MALDI-MS and LC/ESIMS analyses. In the uncatalyzed refolding, a total of 12 disulfide bonds out of the 28 theoretical possible cysteine couplings was observed, confirming a nonrandom distribution of native and nonnative disulfide bonds. Under the presence of PDI, only two additional nonnative disulfides were detected. Semiquantitative LC/ESIMS analysis of the distribution of the S-S bridged peptides showed that the most abundant species were equally populated in both the uncatalyzed and the catalyzed process. This paper shows the first structural characterization of the one-disulfide intermediate population formed transiently during the refolding of ribonuclease A in quasi-physiological conditions that mimic those present in the ER lumen. At the early stages of the process, three of the four native disulfides are detected, whereas the Cys26-Cys84 pairing is absent. Most of the nonnative disulfide bonds identified are formed by nearest-neighboring cysteines. The presence of PDI does not significantly alter the distribution of S-S bonds, suggesting that the ensemble of single-disulfide species is formed under thermodynamic control.     

Structure and heterogeneity of the one- and two-disulfide folding intermediates of tick anticoagulant peptide

Tick anticoagulant peptide (TAP) is a factor Xa-specific inhibitor and is structurally homologous to bovine pancreatic trypsin inhibitor (BPTI). The fully reduced TAP refolds spontaneously to form the native structure under a wide variation of redox buffers. The folding intermediates of TAP consist of at least 22 fractions of one-disulfide, two-disulfide, and three-disulfide scrambled isomers. Three species of well-populated one- and two-disulfide intermediates were isolated and structurally characterized. The predominant one-disulfide species contains TAP-(Cys33—Cys55). Two major two-disulfide isomers were TAP-(Cys33—Cys55, Cys15—Cys39) and TAP-(Cys33—Cys55, Cys5—Cys39). Both Cys33—Cys55 and Cys15—Cys39 are native disulfides of TAP. These three species are structural counterparts of BPTI-(Cys30—Cys51), BPTI-(Cys30—Cys51, Cys14—Cys38), and BPTI-(Cys30—Cys51,Cys5—Cys38), which have been shown to be the major intermediates of BPTI folding. In addition, time-course-trapped folding intermediates of TAP, consisting of about 47% one-disulfide species and 30% two-disulfide species, were collectively digested with thermolysin, and fragmented peptides were analyzed by Edman sequencing and mass spectrometry in order to characterize the disulfide-containing peptides. Among the 15 possible single-disulfide pairings of TAP, 10 (2 native and 8 nonnative) were found as structural components of its one- and two-disulfide folding intermediates. The results demonstrate that the major folding intermediates of TAP bear structural homology to those of BPTI. However, the folding pathway of TAP differs from that of BPTI by (a) a higher degree of heterogeneity of one- and two-disulfide intermediates and (b) the presence of three-disulfide scrambled isomers as folding intermediates. Mechanism(s) that may account for these diversities are proposed and discussed.     

Oxidative folding of bovine pancreatic ribonuclease A: insight into the overall catalysis of the refolding pathway by phosphate

The effects of the strong stabilizing anion, phosphate, on the oxidative folding of bovine pancreatic ribonuclease A were examined. Phosphate was found to catalyze several steps involved in the oxidative folding process at pH 8.0 and 25°C, resulting in an increase in the rate of pre-equilibration of unstructured species on the folding pathway. In the presence of 400 mM phosphate, the overall increase in the rate of regeneration of native protein was caused primarily by the increased formation and stabilization of tertiary structure in the nativelike intermediates, des-[40-95] and des-[65-72], involved in the rate-determining step. Based on the regeneration of native protein and the stability of Cys Ala substituted mutant analogs of the des-species, (C40A, C95A) and (C65A, C72A), it is suggested that the primary role of phosphate is to catalyze the overall regeneration of native protein through nonspecific electrostatic and hydrogen-bonding effects on the protein and solvent.     

A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways.

The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.     

Structure of a partially unfolded form of Escherichia coli dihydrofolate reductase provides insight into its folding pathway

Proteins frequently fold via folding intermediates that correspond to local minima on the conformational energy landscape. Probing the structure of the partially unfolded forms in equilibrium under native conditions can provide insight into the properties of folding intermediates. To elucidate the structures of folding intermediates of Escherichia coli dihydrofolate reductase (DHFR), we investigated transient partial unfolding of DHFR under native conditions. We probed the structure of a high‐energy conformation susceptible to proteolysis (cleavable form) using native‐state proteolysis. The free energy for unfolding to the cleavable form is clearly less than that for global unfolding. The dependence of the free energy on urea concentration (m‐value) also confirmed that the cleavable form is a partially unfolded form. By assessing the effect of mutations on the stability of the partially unfolded form, we found that native contacts in a hydrophobic cluster formed by the F‐G and Met‐20 loops on one face of the central β‐sheet are mostly lost in the partially unfolded form. Also, the folded region of the partially unfolded form is likely to have some degree of structural heterogeneity. The structure of the partially unfolded form is fully consistent with spectroscopic properties of the near‐native kinetic intermediate observed in previous folding studies of DHFR. The findings suggest that the last step of the folding of DHFR involves organization in the structure of two large loops, the F‐G and Met‐20 loops, which is coupled with compaction of the rest of the protein.     

Mutational analysis of the BPTI folding pathway: I. Effects of aromatic-->leucine substitutions on the distribution of folding intermediates.

The roles of aromatic residues in determining the folding pathway of bovine pancreatic trypsin inhibitor (BPTI) were analyzed mutationally by examining the distribution of disulfide-bonded intermediates that accumulated during the refolding of protein variants in which tyrosine or phenylalanine residues were individually replaced with leucine. The eight substitutions examined all caused significant changes in the intermediate distribution. In some cases, the major effect was to decrease the accumulation of intermediates containing two of the three disulfides found in the native protein, without affecting the distribution of earlier intermediates. Other substitutions, however, led to much more random distributions of the intermediates containing only one disulfide. These results indicate that the individual residues making up the hydrophobic core of the native protein make clearly distinguishable contributions to conformation and stability early in folding: The early distribution of intermediates does not appear to be determined by a general hydrophobic collapse. The effects of the substitutions were generally consistent with the structures of the major intermediates determined by NMR studies of analogs, confirming that the distribution of disulfide-bonded species is determined by stabilizing interactions within the ordered regions of the intermediates. The plasticity of the BPTI folding pathway implied by these results can be described using conformational funnels to illustrate the degree to which conformational entropy is lost at different stages in the folding of the wild-type and mutant proteins.     

Recombinant human insulin: X simplification of folding of the biotechnological precursor of recombinant human insulin

The folding of biotechnological precursor of the human insulin precursor was carried out from solubilized inclusion bodies without a preliminary oxidizing or reducing its Cys residues. The inclusion bodies were dissolved in 8 M urea with the addition of 10 mM 2-mercaptoethanol. Hydrophobic cell components were removed from the solution by passing through a neutral weakly hydrophobic sorbent, the solution was five times diluted and refolded upon addition of 0.3 mM cystine for initiation of disulfide rearrangement. The presence of nucleic acids and cell protein impurities does not affect the folding efficiency. The resulting precursor of folded human insulin was purified by metal-chelate affinity chromatography and converted into insulin by two-stage enzymatic cleavage.     

Simulation and experiment conspire to reveal cryptic intermediates and a slide from the nucleation-condensation to framework mechanism of folding

There is a change from three-state to two-state kinetics of folding across the homeodomain superfamily of proteins as the mechanism slides from framework to nucleation-condensation. The tendency for framework folding in this family correlates with inherent helical propensity. The cellular myeloblastis protein (c-Myb) falls in the mechanistic transition region. An earlier, preliminary report of protein engineering experiments and molecular dynamics simulations (MD) showed that the folding mechanism for this protein has aspects of both the nucleation-condensation and framework models. In the more in-depth analysis of the MD trajectories presented here, we find that folding may be attributed to both of these mechanisms in different regions of the protein. The folding of the loop, middle helix, and turn is best described by nucleation-condensation, whereas folding of the N and C-terminal helices may be described by the framework model. Experimentally, c-Myb folds by apparent two-state kinetics, but the MD simulations predict that the kinetics hide a high-energy intermediate. We stabilized this hypothetical folding intermediate by deleting a residue (P174) in the loop between its second and third helices, and the mutant intermediate is long-lived in the simulations. Equilibrium and kinetic experiments demonstrate that folding of the DeltaP174 mutant is indeed three-state. The presence and shape of the intermediate observed in the simulations were confirmed by small angle X-ray scattering experiments.     

Immunochemical pulsed-labeling characterization of intermediates during hen lysozyme oxidative folding

Previous studies have shown that reduced hen egg white lysozyme refolds and oxidizes according to a linear model, in which the number of disulfide bonds increases sequentially. In this study, we describe the kinetics of native tertiary structure formation during the oxidative-renaturation of reduced hen egg white lysozyme, as monitored using an immunochemical pulsed-labeling method based on enzyme-linked immunosorbent assay (ELISA) in conjunction with two monoclonal antibodies (mAb). Each of these antibodies recognizes a separate face of the native lysozyme surface and, more importantly, each epitope is composed of discontinuous regions of the polypeptide chain. Renaturation kinetics were studied under the same refolding conditions as previous investigations of the kinetics of the regain of far-UV CD, fluorescence, enzymatic activity, and disulfide bonds. Comparison of our results with the results from those studies showed that the immunoreactivity (i.e., the native fold) of the alpha-domain appeared in intermediates containing two SS bonds only (C6-C127 and C30-C115), while the immunoreactivity of the beta-domain appeared together with the formation of the third SS bond (C64-C80). Thus, the alpha-domain folds before the beta-domain during the oxidative folding of reduced lysozyme.     

Peptide models of four possible insulin folding intermediates with two disulfides

The single-chain insulin (PIP) can spontaneously fold into native structure through preferred kinetic intermediates. During refolding, pairing of the first disulfide A20-B19 is highly specific, whereas pairing of the second disulfide is likely random because two two-disulfide intermediates have been trapped. To get more details of pairing property of the second disulfide, four model peptides of possible folding intermediates with two disulfides were prepared by protein engineering, and their properties were analyzed. The four model peptides were named [A20-B19, A7-B7]PIP, [A20-B19, A6-B7]PIP, [A20-B19, A6-A11]PIP, and [A20-B19, A7-A11]PIP according to their remaining disulfides. The four model peptides all adopt partially folded structure with moderate conformational differences. In redox buffer, the disulfides of the model peptides are more easily reduced than those of the wild-type PIP. During in vitro refolding, the reduced model peptides share similar relative folding rates but different folding yields: The refolding efficiency of the reduced [A20-B19, A7-A11]PIP is about threefold lower than that of the other three peptides. The present results indicate that the folding intermediates corresponding to the present model peptides all adopt partially folded conformation, and can be formed during PIP refolding, but the chance of forming the intermediate with disulfide [A20-B19, A7-A11] is much lower than that of forming the other three intermediates.     

Protein folding, misfolding, and refolding of therapeutic proteins

Substantial progress has been made towards understanding the folding mechanisms of proteins in vitro and in vivo even though the general rules governing such folding events remain unknown. This paper reviews current folding models along with experimental approaches used to elucidate the folding pathways. Protein misfolding is discussed in relation to disease states, such as amyloidosis, and the recent findings on the mechanism of converting normally soluble proteins into amyloid fibrils through the formation of intermediates provide an insight into understanding the pathogenesis of amyloid formation and possible clues for the development of therapeutic treatments. Finally, some commonly adopted refolding strategies developed over the past decade are summarized.     

Absence of a stable intermediate on the folding pathway of protein A.

The B-domain of protein A has one of the simplest protein topologies, a three-helix bundle. Its folding has been studied as a model for elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C-terminal helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped-flow circular dichroism and NMR hydrogen-deuterium exchange pulse labeling. Folding of the entire molecule is essentially complete within the 6 ms dead time of the quench-flow apparatus, indicating that the intermediate, if formed, progresses rapidly to the final folded state. Site-directed mutagenesis of the isoleucine residue at position 16 was used to generate a variant protein containing tryptophan (the 116 W mutant). The formation of the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unfavorable steric interactions of the bulky tryptophan side chain in the folded state. The 116 W mutant refolds completely within the dead time of a stopped-flow fluorescence experiment. No partly folded intermediate could be detected by either kinetic or equilibrium measurements. Studies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, its stability appears to be marginal (of the order of 1/2 kT) and it could not be an obligatory intermediate on a defined folding pathway. These results explicitly demonstrate that the protein A B domain folds extremely rapidly by an apparent two-state mechanism without formation of stable partly folded intermediates. Similar mechanisms may also be involved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.     

Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates.

The Saccharomyces cerevisiae non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The N-terminal prion domain (PrD) of Ure2 is required for prion activity in vivo and amyloid formation in vitro. However, the molecular mechanism of the prion-like activity remains obscure. Here we measure the kinetics of folding of Ure2 and two N-terminal variants that lack all or part of the PrD. The kinetic folding behaviour of the three proteins is identical, indicating that the PrD does not change the stability, rates of folding or folding pathway of Ure2. Both unfolding and refolding kinetics are multiphasic. An intermediate is populated during unfolding at high denaturant concentrations resulting in the appearance of an unfolding burst phase and "roll-over" in the denaturant dependence of the unfolding rate constants. During refolding the appearance of a burst phase indicates formation of an intermediate during the dead-time of stopped-flow mixing. A further fast phase shows second-order kinetics, indicating formation of a dimeric intermediate. Regain of native-like fluorescence displays a distinct lag due to population of this on-pathway dimeric intermediate. Double-jump experiments indicate that isomerisation of Pro166, which is cis in the native state, occurs late in refolding after regain of native-like fluorescence. During protein refolding there is kinetic partitioning between productive folding via the dimeric intermediate and a non-productive side reaction via an aggregation prone monomeric intermediate. In the light of this and other studies, schemes for folding, aggregation and prion formation are proposed.     

Kinetic evidence of an on-pathway intermediate in the folding of lysozyme

By means of a kinetic test, it was demonstrated that one of the folding intermediates (Ialpha) of hen lysozyme with alpha-domain folded and beta-domain unfolded is on the folding pathway under the classical definition. Ialpha folds to the native (N) state directly (unfolded (U) <==> Ialpha <==> N) without having to unfold to U and then refold to N through alternative folding pathways as in Ialpha <==> U <==> N.