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.     

In vitro refolding/unfolding pathways of amphioxus insulin-like peptide: implications for folding behavior of insulin family proteins

Amphioxus insulin-like peptide (AILP) belongs to the insulin superfamily and is proposed as the common ancestor of insulin and insulin-like growth factor 1. Herein, the studies on oxidative refolding and reductive unfolding of AILP are reported. During the refolding process, four major intermediates, P1, P2, P3, and P4, were captured, which were almost identical to those intermediates, U1, U2, U3, and U4, captured during the AILP unfolding process. P4 (U4) has the native disulfide A20-B19; P1 (U1), P2 (U2), and P3 (U3) have two disulfide bonds, which include A20-B19. Based on the analysis of the time course distribution and properties of the intermediates, we proposed that fully reduced AILP refolded through 1SS, 2SS, and 3SS intermediate stages to the native form; native AILP unfolded through 2SS and 1SS intermediate stages to the full reduced form. A schematic flow chart of major oxidative refolding and reductive unfolding pathways of AILP was proposed. Implication for the folding behavior of insulin family proteins was discussed. There may be seen three common folding features in the insulin superfamily: 1) A20-B19 disulfide is most important and formed during the initial stage of folding process; 2) the second disulfide is nonspecifically formed, which then rearranged to native disulfide; 3) in vitro refolding and unfolding pathways may share some common folding intermediates but flow in opposite directions. Furthermore, although swap AILP is a thermodynamically stable final product, a refolding study of swap AILP demonstrated that it is also a productive intermediate of native AILP during refolding.     

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.     

蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究,蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中,大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠,这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥,因为后者仅仅是进行链内二硫键重排的化学反应所必需,与控制肽链折叠无直接关系。3根据对BPTI的研究,二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用,既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明,天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成,更具有普遍意义。4在氧化重折叠的早期,二硫键的形成基本上是一个随机过程,随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外,在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述,在折叠的后期,二硫键的形成决定于折叠中间体的构象,类天然、有柔性的结构有利于天然二硫键形成和正确折叠,具有这类结构的分子为有效的折叠中间体,最终都能转变为天然产物;而无效折叠中间体往往具有稳定的结构,使巯基、二硫键内埋妨碍二硫键重排,并因能垒的障碍不利于进一步折叠。因此,降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则,实验证明,碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外,这里还就氧化重折叠的基础和应用研究的前景进行了讨论。     

胰岛素折叠、结合与稳定性的核磁共振研究(英)

Insulin is one of the most important hormonal regulators of metabolism. Since the diabetes patients increase dramatically, the chemical properties, biological and physiological effects of insulin had been extensively studied. In last decade the development of NMR technique allowed us to determine the solution structures of insulin and its variety mutants in various conditions, so that the knowledge of folding, binding and stability of insulin in solution have been largely increased. The solution structure of insulin monomers is essentially identical to those of insulin monomers within the dimer and bexamer as determined by X-ray diffraction. The studies of insulin mutants at the putative residues for receptor binding explored the possible conformational change and fitting between insulin and its receptor. The systematical studies of disulfide paring coupled insulin folding intermediates revealed that in spite of the conformational variety of the intermediates, one structural feature is always remained: a “native-like B chain super-secondary structure“, which consists of B9-B19 helix with adjoining B23-B26 segment folded back against the central segment of B chain, an internal cystine A20-B19 disulfide bridge and a short a-helix at C-terminal of A chain linked. The “super-secondary structure“ might be the “folding nucleus“ in insulin folding mechanism. Cystine A20-B19 is the most important one among three disulfides to stabilize the nascent polypeptide in early stage of the folding. The NMR structure of C. elegans insulin-like peptide resembles that of human insulin and the peptide interacts with human insulin receptor. Other members of insulin superfamily adopt the “insulin fold“ mostly. The structural study of insulin-insulin receptor complex, that of C elegans and other invertebrate insulin-like peptide, insulin fibril study and protein disulfide isomerase (PDI) assistant proinsulin folding study will be new topics in future to get insight into folding, binding, stability, evolution and fibrillation of insulin in detail.     

In vitro folding of methionine‐arginine human lyspro‐proinsulin S‐sulfonate—Disulfide formation pathways and factors controlling yield

We investigated the in vitro folding of an oxidized proinsulin (methionine‐arginine human lyspro‐proinsulin S‐sulfonate), using cysteine as a reducing agent at 5°C and high pH (10.5–11). Folding intermediates were detected and characterized by means of matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS), reversed‐phase chromatography (RPC), size‐exclusion chromatography, and gel electrophoresis. The folding kinetics and yield depended on the protein and cysteine concentrations. RPC coupled with MALDI‐MS analyses indicated a sequential formation of intermediates with one, two, and three disulfide bonds. The MALDI‐MS analysis of Glu‐C digested, purified intermediates indicated that an intra‐A‐chain disulfide bond formed first among A6, A7, and A11. Various non‐native intra‐A (A20 with A6, A7, or A11), intra‐B (between B7 and B19), and inter‐A‐B disulfide bonds were observed in the intermediates with two disulfide bonds. The intermediates with three disulfide bonds had mainly the non‐native intra‐A and intra‐B bonds. At a cysteine‐to‐proinsulin‐SH ratio of 3.5, all intermediates with the non‐native disulfide bonds were converted to properly folded proinsulin via disulfide bond reshuffling, which was the slowest step. Aggregation via the formation of intermolecular disulfide bonds of early intermediates was the major cause of yield loss. At a higher cysteine‐to‐proinsulin‐SH ratio, some intermediates and folded MR‐KPB‐hPI were reduced to proteins with thiolate anions, which caused unfolding and even more yield loss than what resulted from aggregation of the early intermediates. Reducing protein concentration, while keeping an optimal cysteine‐to‐protein ratio, can improve folding yield significantly. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Pathway of oxidative folding of secretory leucocyte protease inhibitor: an 8-disulfide protein exhibits a unique mechanism of folding

Secretory leucocyte protease inhibitor (SLPI) is a 107-amino acid protein with a high density of disulfide pairing (eight). The mechanism of oxidative folding of reduced and denatured SLPI has been investigated here. Despite an exceedingly large number of possible folding intermediates ( approximately 46 million disulfide isomers) and their potential to complicate the refolding process, oxidative folding of SLPI turns out to be surprisingly simple and efficient. Complete oxidative folding and a near-quantitative recovery of the native SLPI can be achieved in a simple buffer solution using air oxidation without any supplementing thiol catalyst or redox agent, a phenomenon that has not yet been observed with other disulfide proteins. Because of the heterogeneity and extensive overlapping of folding intermediates, identification of the predominant intermediate was unfeasible. Nonetheless, studies of reductive unfolding of native SLPI and oxidative folding of a six-disulfide variant of SLPI enable us to propose an underlying mechanism accounting for the unique folding efficiency of SLPI in the absence of a redox agent. Our studies indicate that oxidative folding of SLPI undergoes heterogeneous populations of one-, two-, three-, four-, five-, six-, and seven-disulfide isomers, including two nativelike isomers, SLPI-6A and SLPI-7A, as transient intermediates. Formation of the last two native disulfide bonds leading to the conversion of SLPI-6A --> SLPI-7A --> N-SLPI is relatively slow and represents the final stage of oxidative folding. Most importantly, free cysteines of SLPI-6A and SLPI-7A also act as a thiol catalyst in promoting the disulfide shuffling of diverse non-native intermediates accumulated along the folding pathway. This explains why a near-quantitative recovery of N-SLPI can be achieved in the absence of any thiol catalyst and redox agent. Properties of SLPI-6A and SLPI-7A were investigated and compared to those of other documented kinetic intermediates of oxidative folding. The correlation between the mechanism of SLPI folding and the three-dimensional structure of SLPI is also elaborated.     

Putative disulfide-forming pathway of porcine insulin precursor during its refolding in vitro

Although the structure of insulin has been well studied, the formation pathway of the three disulfide bridges during the refolding of insulin precursor is ambiguous. Here, we reported the in vitro disulfide-forming pathway of a recombinant porcine insulin precursor (PIP). In redox buffer containing L-arginine, the yield of native PIP from fully reduced/denatured PIP can reach 85%. The refolding process was quenched at different time points, and three distinct intermediates, including one with one disulfide linkage and two with two disulfide bridges, have been captured and characterized. An intra-A disulfide bridge was found in the former but not in the latter. The two intermediates with two disulfide bridges contain the common A20-B19 disulfide linkage and another inter-AB one. Based on the time-dependent formation and distribution of disulfide pairs in the trapped intermediates, two different forming pathways of disulfide bonds in the refolding process of PIP in vitro have been proposed. The first one involves the rapid formation of the intra-A disulfide bond, followed by the slower formation of one of the inter-AB disulfide bonds and then the pairing of the remaining cysteines to complete the refolding of PIP. The second pathway begins first with the formation of the A20-B19 disulfide bridge, followed immediately by another inter-AB one, possibly nonnative. The nonnative two-disulfide intermediates may then slowly rearrange between CysA6, CysA7, CysA11, and CysB7, until the native disulfide bond A6-A11 or A7-B7 is formed to complete the refolding of PIP. The proposed refolding behavior of PIP is compared with that of IGF-I and discussed.     

Unfolding of human proinsulin. Intermediates and possible role of its C-peptide in folding/unfolding.

We have investigated the in vitro refolding process of human proinsulin (HPI) and an artificial mini-C derivative of HPI (porcine insulin precursor, PIP), and found that they have significantly different disulfide-formation pathways. HPI and PIP differ in their amino acid sequences due to the presence of the C-peptide linker found in HPI, therefore suggesting that the C-peptide linker may be responsible for the observed difference in folding behaviour. However, the manner in which the C-peptide contributes to this difference is still unknown. We have used both the disulfide scrambling method and a redox-equilibrium assay to assess the stability of the disulfide bridges. The results show that disulfide reshuffling is easier to induce in HPI than in PIP by the addition of thiol reagent. Thus, the C-peptide may affect the unique folding pathway of HPI by allowing the disulfide bonds of HPI to be easily accessible. The detailed processes of HPI unfolding by reduction of its disulfide bonds and by disulfide scrambling methods were also investigated. In the reductive unfolding process no accumulation of intermediates was detected. In the process of unfolding by disulfide scrambling, HPI gradually rearranged its disulfide bonds to form three major isomers G1, G2 and G3. The most abundant isomer, G1, contains the B7-B19 disulfide bridge. Based on far-UV CD spectra, native gel analysis and cleavage by endoproteinase V8, the G1 isomer has been shown to resemble the intermediate P4 found in the refolding process of HPI. Finally, the major isomer G1 is allowed to refold to native protein HPI by disulfide rearrangement, which indicates that a similar molecular mechanism may exist for the unfolding and refolding process of HPI.     

Deciphering the Hidden Informational Content of Protein Sequences: FOLDABILITY OF PROINSULIN HINGES ON A FLEXIBLE ARM THAT IS DISPENSABLE IN THE MATURE HORMONE*

Protein sequences encode both structure and foldability. Whereas the interrelationship of sequence and structure has been extensively investigated, the origins of folding efficiency are enigmatic. We demonstrate that the folding of proinsulin requires a flexible N-terminal hydrophobic residue that is dispensable for the structure, activity, and stability of the mature hormone. This residue (Phe^B1 in placental mammals) is variably positioned within crystal structures and exhibits ¹H NMR motional narrowing in solution. Despite such flexibility, its deletion impaired insulin chain combination and led in cell culture to formation of non-native disulfide isomers with impaired secretion of the variant proinsulin. Cellular folding and secretion were maintained by hydrophobic substitutions at B1 but markedly perturbed by polar or charged side chains. We propose that, during folding, a hydrophobic side chain at B1 anchors transient long-range interactions by a flexible N-terminal arm (residues B1–B8) to mediate kinetic or thermodynamic partitioning among disulfide intermediates. Evidence for the overall contribution of the arm to folding was obtained by alanine scanning mutagenesis. Together, our findings demonstrate that efficient folding of proinsulin requires N-terminal sequences that are dispensable in the native state. Such arm-dependent folding can be abrogated by mutations associated with β-cell dysfunction and neonatal diabetes mellitus.     

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.     

Crystal Structure of a ��Nonfoldable�� Insulin: IMPAIRED FOLDING EFFICIENCY DESPITE NATIVE ACTIVITY*

Protein evolution is constrained by folding efficiency (“foldability”) and the implicit threat of toxic misfolding. A model is provided by proinsulin, whose misfolding is associated with β-cell dysfunction and diabetes mellitus. An insulin analogue containing a subtle core substitution (Leu^A16 → Val) is biologically active, and its crystal structure recapitulates that of the wild-type protein. As a seeming paradox, however, Val^A16 blocks both insulin chain combination and the in vitro refolding of proinsulin. Disulfide pairing in mammalian cell culture is likewise inefficient, leading to misfolding, endoplasmic reticular stress, and proteosome-mediated degradation. Val^A16 destabilizes the native state and so presumably perturbs a partial fold that directs initial disulfide pairing. Substitutions elsewhere in the core similarly destabilize the native state but, unlike Val^A16, preserve folding efficiency. We propose that Leu^A16 stabilizes nonlocal interactions between nascent α-helices in the A- and B-domains to facilitate initial pairing of Cys^A20 and Cys^B19, thus surmounting their wide separation in sequence. Although Val^A16 is likely to destabilize this proto-core, its structural effects are mitigated once folding is achieved. Classical studies of insulin chain combination in vitro have illuminated the impact of off-pathway reactions on the efficiency of native disulfide pairing. The capability of a polypeptide sequence to fold within the endoplasmic reticulum may likewise be influenced by kinetic or thermodynamic partitioning among on- and off-pathway disulfide intermediates. The properties of [Val^A16]insulin and [Val^A16]proinsulin demonstrate that essential contributions of conserved residues to folding may be inapparent once the native state is achieved.     

The nature of protein folding pathways: The classical versus the new view

Summary Pulsed hydrogen exchange and other studies of the kinetic refolding pathways of several small proteins have established that folding intermediates with native-like secondary structures are well populated, but these studies have also shown that the folding kinetics are not well synchronized. Older studies of the kinetics of formation of the native protein, monitored by optical probes, indicate that the folding kinetics should be synchronized. The model commonly used in these studies is the simple sequential model, which postulates a unique folding pathway with defined and sequential intermediates. Theories of the folding process and Monte Carlo simulations of folding suggest that neither the folding pathway nor the set of folding intermediates is unique, and that folding intermediates accumulate because of kinetic traps caused by partial misfolding. Recent experiments with cytochrome c lend support to this new view of folding pathways. These different views of the folding process are discussed. Misfolding and consequent slowing down of the folding process as a result of cis-trans isomerization about prolyl peptide bonds in the unfolded protein are well known; isomerization occurs before refolding is initiated. The occurrence of equilibrium intermediates on the kinetic folding pathways of some proteins, such as -lactalbumin and apomyoglobin, argues that these intermediates are not caused by kinetic traps but rather are stable intermediates under certain conditions, and this conclusion is consistent with a sequential model of folding. Folding reactions with successive kinetic intermediates, in which late intermediates are more highly folded than early intermediates, indicate that folding is hierarchical. New experiments that test the predictions of the classical and the new views are needed.     

Disulfide exchange folding of insulin-like growth factor I.

The disulfide exchange folding properties of insulin-like growth factor I (IGF-I) have been analyzed in a redox buffer containing reduced (10 mM) and oxidized (1 mM) glutathione. Under these conditions, the 3 disulfide bridges of the 70 amino acid peptide were not quantitatively formed. Instead, five major forms of IGF-I were detected, and these components were concluded to be in equilibrium as their relative amounts were similar starting from either reduced, native, or a mismatched variant of IGF-I containing two non-native disulfides. The different components in the mixtures were trapped by thiol alkylation using vinylpyridine and subsequently isolated by reverse-phase HPLC. The purified variants were further characterized using plasma desorption mass spectrometry and peptide mapping. Two of the five different forms were identified as native and mismatched IGF-I. One form was a variant with only one disulfide bond, and the other two major components had two disulfides formed. In a separate experiment, early refolding intermediates were trapped by pyridylethylation after only 90 s of refolding in the glutathione buffer, starting from reduced IGF-I. The intermediates were identical to the components observed at equilibrium, but at different relative concentrations. On the basis of the disulfide bond patterns of the different components in the equilibrium mixtures, we conclude that the disulfide between cysteines-47 and -52 in IGF-I is an unfavorable high-energy bond that may exist in the native molecule in a strained configuration.     

The oxidative refolding of hen lysozyme and its catalysis by protein disulfide isomerase.

The oxidative refolding of hen lysozyme has been studied by a variety of time-resolved biophysical methods in conjunction with analysis of folding intermediates using reverse-phase HPLC. In order to achieve this, refolding conditions were designed to reduce aggregation during the early stages of the folding reaction. A complex ensemble of relatively unstructured intermediates with on average two disulfide bonds is formed rapidly from the fully reduced protein after initiation of folding. Following structural collapse, the majority of molecules slowly form the four-disulfide-containing fully native protein via rearrangement of a highly native-like, kinetically trapped intermediate, des-[76-94], although a significant population (approximately 30%) appears to fold more quickly via other three-disulfide intermediates. The folding catalyst PDI increases dramatically both yields and rates of lysozyme refolding, largely by facilitating the conversion of des-[76-94] to the native state. This suggests that acceleration of the folding rate may be an important factor in avoiding aggregation in the intracellular environment.     

Study of a major intermediate in the oxidative folding of leech carboxypeptidase inhibitor: contribution of the fourth disulfide bond

The oxidative folding pathway of leech carboxypeptidase inhibitor (LCI; four disulfide bonds) proceeds through the formation of two major intermediates (III-A and III-B) that contain three native disulfide bonds and act as strong kinetic traps in the folding process. The III-B intermediate lacks the Cys19-Cys43 disulfide bond that links the beta-sheet core with the alpha-helix in wild-type LCI. Here, an analog of this intermediate was constructed by replacing Cys19 and Cys43 with alanine residues. Its oxidative folding follows a rapid sequential flow through one, two, and three disulfide species to reach the native form; the low accumulation of two disulfide intermediates and three disulfide (scrambled) isomers accounts for a highly efficient reaction. The three-dimensional structure of this analog, alone and in complex with carboxypeptidase A (CPA), was determined by X-ray crystallography at 2.2A resolution. Its overall structure is very similar to that of wild-type LCI, although the residues in the region adjacent to the mutation sites show an increased flexibility, which is strongly reduced upon binding to CPA. The structure of the complex also demonstrates that the analog and the wild-type LCI bind to the enzyme in the same manner, as expected by their inhibitory capabilities, which were similar for all enzymes tested. Equilibrium unfolding experiments showed that this mutant is destabilized by approximately 1.5 kcal mol(-1) (40%) relative to the wild-type protein. Together, the data indicate that the fourth disulfide bond provides LCI with both high stability and structural specificity.     

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.     

Disulfide-linked bovine beta-lactoglobulin dimers fold slowly, navigating a glassy folding landscape

To gain insight into the folding of large proteins, we constructed a bovine beta-lactoglobulin (beta-lg) dimeric mutant, A34C/C121A beta-lg. In the mutant, a free thiol group of wild-type beta-lg at Cys121 was removed and two beta-lg molecules were linked by a disulfide bridge through Cys34 created at the dimer's interface. Under strongly native conditions at low concentrations of urea, the refolding yield of A34C/C121A beta-lg was low when monitored by heteronuclear NMR spectroscopy. However, under marginally native conditions, the yield improved notably, although the refolding was still slow. H-D exchange pulse labeling monitored using heteronuclear NMR spectroscopy indicated that A34C/C121A beta-lg forms a folding intermediate similar to monomeric C121A beta-lg in spite of its slow folding. These results indicate that the rapid formation of folding intermediates driven by local interactions occurs in a manner independent of the molecular size and that, if the non-native interactions are too strong, the kinetic trap is set, leading to a glasslike misfolded state. The results suggest the important roles of marginal stability and pathways in making the folding of large proteins possible.     

Intimate view of a kinetic protein folding intermediate: residue-resolved structure, interactions, stability, folding and unfolding rates, homogeneity

A cytochrome c kinetic folding intermediate was studied by hydrogen exchange (HX) pulse labeling. Advances in the technique and analysis made it possible to define the structured and unstructured regions, equilibrium stability, and kinetic opening and closing rates, all at an amino acid-resolved level. The entire N-terminal and C-terminal helices are formed and docked together at their normal native positions. They fray in both directions from the interaction region, due to a progression in both unfolding and refolding rates, leading to the surprising suggestion that helix propagation may proceed very slowly in the condensed milieu. Several native-like beta turns are formed. Some residues in the segment that will form the native 60s helix are protected but others are not, suggesting energy minimization to some locally non-native conformation in the transient intermediate. All other regions are unprotected, presumably dynamically disordered. The intermediate resembles a partially constructed native state. It is early, on-pathway, and all of the refolding molecules pass through it. These and related results consistently point to distinct, homogeneous, native-like intermediates in a stepwise sequential pathway, guided by the same factors that determine the native structure. Previous pulse labeling efforts have always assumed EX2 exchange during the labeling pulse, often leading to the suggestion of heterogeneous intermediates in alternative parallel pathways. The present work reveals a dominant role for EX1 exchange in the high pH labeling pulse, which will mimic heterogeneous behavior when EX2 exchange is assumed. The general problem of homogeneous versus heterogeneous intermediates and pathways is discussed.