Non-native Isomers Research Articles

Cysteine-based protein folding modulators for trapping intermediates and misfolded forms.

Folding is a key process to form functional conformations of proteins. Folding via on-pathway intermediates leads to the formation of native structures, while folding through off-pathways affords non-native and disease-causing forms. Trapping folding intermediates and misfolded forms is important for investigating folding mechanisms and disease-related biological properties of the misfolded proteins. We developed cysteine-containing dipeptides conjugated with amino acids possessing mono- and diamino-groups. In oxidative protein folding involving disulfide-bond formation, the addition of cysteine and oxidized glutathione readily promoted the folding to afford native forms. In contrast, despite the acceleration of disulfide-bond formation, non-native isomers formed in significantly increased yields upon the addition of the dipeptides. This study provides a molecular design of cysteine-based protein-folding modulators that afford proteins adopting non-native conformations through intermolecular disulfide-bond formation. Because of the intrinsic reversibility of the disulfide bonds upon redox reactions, the disulfide bond-based approach demonstrated here is expected to lead to the development of reversible methodologies for trapping transient and misfolded forms by intermolecular disulfide bond formation and restarting the folding processes of the trapped forms by subsequent cleavage of the intermolecular disulfide bonds.

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the oxidative folding of Cox19, a twin CX9C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second helical CX9C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive folding and mimic the Mia40 folding template mechanism. The Mia40 role is to funnel a rough folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.

Handling a tricycle: Orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin

Gurmarin is a 35 amino acid peptide with three disulfide bridges in an inhibitor cystine knot. It is found in the plant Gymnema sylvestre, and has been identified as a sweet taste inhibitor in rodents. In this article we provide an efficient route for the synthesis of gurmarin by a controlled random oxidation strategy. We compared two oxidation procedures to form the three disulfide bridges. In the first, based on random oxidation, reduced gurmarin was synthesized using trityl for cysteine protection, and oxidized for 48h in a Tris–HCl buffer containing cystamine and reduced glutathione to facilitate disulfide scrambling. The second was based on step-wise deprotection followed by oxidation in which the cysteine pairs are orthogonally protected with tert-Butylthio, trityl and acetamidomethyl. To verify that the native gurmarin oxidation product was obtained, thermolysin cleavage was used. Cleavage of random oxidized gurmarin showed two possible disulfide combinations; the native and a non-native gurmarin disulfide isomer. The non-native isomer was therefore synthesized using the orthogonal deprotection-oxidation strategy and the native and the non-native gurmarin isomers were analyzed using UPLC. It was found that the random oxidation procedure leads to native gurmarin in high yield. Thus, the synthetic route was simple and significantly more efficient than previously reported syntheses of gurmarin and other cysteine rich peptides. Importantly, native gurmarin was obtained by random oxidation, which was confirmed by a synthetic approach for the first time.

Synthesis and Immune Response of Non-native Isomers of Vascular Endothelial Growth Factor

Native proteins often lack immunogenicity and thus limit vaccine and mAb development. We described here a unique method to enhance the immunogenicity of native proteins. This is achieved by creating non-native isomers of disulfide proteins (X-isomers) using the method of disulfide scrambling. X-isomers have the potential to be developed as vaccines and effective immunogens, as they are capable of breaking the immune tolerance and eliciting antibodies that cross-react with the native protein. In this report, we describe production of X-isomers of vascular endothelial growth factor (X-VEGF). The aim is to develop X-VEGF for cancer immunotherapy targeting reduction of VEGF. The production of mouse X-VEGF is achieved by expressing the short version of VEGF (1-110) commonly shared by all VEGF isoforms, with two Cys --> Ala mutations at Cys(51) and Cys(60) to generate R-VEGF(110) (R stands for fully reduced). R-VEGF(110) was then allowed to undergo oxidative folding in the absence of denaturant to form N-VEGF(110) (N stands for native) or in the presence of denaturant to generate five fractions of X-VEGF(110) isomers. While N-VEGF(110) exhibits only marginal immunogenicity in mice, all five fractions of X-VEGF(110) isomers were shown to elicit high titers of antibodies that cross-react with N-VEGF(110). In sera of immunized mice, the amounts of anti-N-VEGF antibodies elicited by X-VEGF(110) isomers range from 54 to 186 mug/mL, which are compatible with or greater than the concentration required for effective therapy using anti-VEGF MAbs. The underlying mechanism of enhanced immunogenicity of X-VEGF(110) is investigated and elaborated. These data suggest that X-VEGF(110) isomers are potential compounds in developing active immunotherapy for treatment of VEGFR bearing tumors and the wet form of age-related macular degeneration.

Woes of proline: A cautionary kinetic tale

Woes of proline: A cautionary kinetic tale

Fully oxidized scrambled isomers are essential and predominant folding intermediates of cardiotoxin-III

Scrambled isomers (X-isomers) are fully oxidized, non-native isomers of disulfide proteins. They have been shown to represent important intermediates along the pathway of oxidative folding of numerous disulfide proteins. A simple method to assess whether X-isomers present as folding intermediate is to conduct oxidative folding of fully reduced protein in the alkaline buffer alone without any supplementing thiol catalyst or redox agent. Cardiotoxin-III (CTX-III) contains 60 amino acids and four disulfide bonds. The mechanism of oxidative folding of CTX-III has been systematically characterized here by analysis of the acid trapped folding intermediates. Folding of CTX-III was shown to proceed sequentially through 1-disulfide, 2-disulfide, 3-disulfide and 4-disulfide (scrambled) isomers as folding intermediates to reach the native structure. When folding of CTX-III was performed in the buffer alone, more than 97% of the protein was trapped as 4-disulfide X-isomers, unable to convert to the native structure due to the absence of thiol catalyst. In the presence of thiol catalyst (GSH) or redox agents (GSH/GSSG), the recovery of native CTX-III was 80–85%. These results demonstrate that X-isomers play an essential and predominant role in the oxidative folding of CTX-III.

Conformational impurity of disulfide proteins: Detection, quantification, and properties

The conformations of native proteins are in principle, and in most cases, dictated by the law of thermodynamics. Accordingly, a native protein must always exist in equilibrium with a minor concentration of nonnative (denatured) conformational isomers even at nondenaturing conditions. The presence of an infinitesimal quantity of nonnative conformational isomers at physiological conditions is biologically relevant due to their propensity to aggregate, which is an underlying cause of many neurodegenerative diseases. However, their detection and quantification are inherently difficult. In this article, we describe a simple strategy using the technique of disulfide scrambling to identify and quantify such minute concentrations of nonnative isomers. It is demonstrated that even for small stable proteins such as epidermal growth factor and hirudin, approximately 1% of heterogeneous nonnative isomers coexist with the native proteins under physiological conditions.

Role of disulfide bonds in folding and activity of leiurotoxin I: just two disulfides suffice.

The aim of this study is to investigate the contribution of each disulfide bond in the folding and function of leiurotoxin I, a short scorpion toxin that blocks small conductance K(+) channels. The structure of leiurotoxin I contains a motif conserved in all scorpion toxins, formed by a helix and a double-stranded beta-sheet and stabilized by three disulfide bridges. We synthesized three analogues, each presenting two alpha-aminobutyric acid (Abu) moieties replacing two bridged cysteine residues: LeTx1 ([Abu 3,21] Leiurotoxin I), LeTx2 ([Abu 8,26] Leiurotoxin I), and LeTx3 ([Abu 12,28] Leiurotoxin I). All three analogues fold into a major product containing two native disulfide bonds, while LeTx3 forms an additional isomer, containing non-native disulfides. In denaturing conditions, analogues LeTx2 and LeTx3 yield non-native isomers, while LeTx1 only forms the isomer with native disulfides. All isomers with native disulfides contain nativelike alpha-helical conformations and bind to synaptosomal membranes with affinities within a log of that shown by the native toxin. By contrast, the non-native LeTx3A analogue exhibits a disordered conformation and a decreased biological potency. Our results indicate that the "CxxxC, CxC" cysteine spacing, conserved in all scorpion toxins and preserved in LeTx1, may play an active role in folding, and that only two native disulfide bonds in leiurotoxin I are sufficient to preserve a nativelike and active conformation. Thus, in the scorpion toxin scaffold, modifications of conserved and interior cysteine residues may permit modulation of function, without significantly affecting folding efficiency and structure.

The thermodynamic stability of insulin disulfides is not affected by the C-domain of insulin-like growth factor 1

Both Insulin and insulin-like growth factor 1 are members of insulin superfamily. They share homologous primary and tertiary structure as well as weakly overlapping biological activity. However, their folding behavior is different: insulin and its recombinant precursor (PIP) fold into one unique tertiary structure, while IGF-1 folds into two disulfides isomers with similar thermodynamic stability. To elucidate the molecular mechanism of their different folding behavior, we prepared a singlechain hybrid of insulin and IGF-1, [B10Glu]Ins/IGF-1(C), and studied its folding behavior compared with that of PIP and IGF-1. We also separated a major non-native disulfides isomer of the hybrid and studied its refolding. The data showed that the C-domain of IGF-1 did not affect the folding thermodynamics of insulin, that is, the primary structure of the hybrid encoded only one thermodynamically stable disulfides linkage. However, the folding kinetics of insulin was affected by the C-domain of IGF-1.

Folding of a disulfide-bonded protein species with free thiol(s): competition between conformational folding and disulfide reshuffling in an intermediate of bovine pancreatic ribonuclease A.

The conformational folding of the nativelike intermediate des-[40-95] on the major oxidative folding pathway of bovine pancreatic ribonuclease A (RNase A) has been examined at various pHs and temperatures in the absence of a redox reagent. Des-[40-95] has three of the four disulfide bonds of native RNase A and lacks the bond between Cys40 and Cys95. This three-disulfide species was unfolded at low pH to inhibit any disulfide reshuffling and was refolded at higher pH, allowing both conformational folding and disulfide-reshuffling reactions to take place. As a result of this competition, 15-85% of des-[40-95], depending on the experimental conditions, undergoes intramolecular disulfide-reshuffling reactions. That portion of the des-[40-95] population which has native isomers of essential proline residues appears to fold faster than the disulfide reaction can occur. However, when the folding is retarded, conceivably by the presence of non-native isomers of essential proline residues, des-[40-95] may reshuffle before completing the conformational folding process. These results enable us to distinguish among current models for the critical structure-forming step in oxidative folding and reveal a new model for coupling proline isomerization to disulfide-bond formation. These experiments also demonstrate that the reshuffling-folding competition assay is a useful tool for detecting structured populations in conformational folding intermediates.

Structure determination of the three disulfide bond isomers of α-conotoxin GI: a model for the role of disulfide bonds in structural stability

The three possible disulfide bonded isomers of α-conotoxin GI have been selectively synthesised and their structures determined by 1H NMR spectroscopy. α-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2–7;3–13), GI(2–13;3–7) and GI(2–3;7–13). The NMR structure for the native isomer GI(2–7;3–13) is of excellent quality, with a backbone pairwise RMSD of 0.16 Å for a family of 35 structures, and comprises primarily a distorted 310helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2–7;3–13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in α-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2–7;3–13)<GI(2–13;3–7)<GI(2–3;7–13). It was found that the peptide bond joining Cys2 to Cys3 in GI(2–3;7–13) is predominantly trans, rather than cis as theoretically predicted. These structural data are used to interpret the varying nAChR binding of the non-native forms.A model for the binding of native GI(2–7;3–13) to the mammalian nAChR is proposed, with an α-subunit binding face made up of Cys2, Asn4, Pro5, Ala6 and Cys7 and a selectivity face, comprised of Arg9 and His10. These two faces orient the molecule between the α and δ subunits of the receptor. The structure of the CCNPAC sequence of the native GI(2–7;3–13) is compared to the structure of the identical sequence from the toxic domain of heat-stable enterotoxins, which forms part of the receptor binding region of the enterotoxins, but which has a different disulfide connectivity.

Trapping of intermediates during the refolding of recombinant human epidermal growth factor (hEGF) by cyanylation, and subsequent structural elucidation by mass spectrometry.

Human epidermal growth factor (hEGF) contains 53 amino acids and three disulfide bonds. The unfolded, reduced hEGF is allowed to refold under mildly alkaline conditions. The folding is quenched at different time points by adjusting the pH to 3.0 with an acetic acid solution of 1-cyano-4-dimethylamino-pyridinium (CDAP) which traps folding intermediates via cyanylation of free sulfhydryl groups. The mixture of cyanylated intermediates is separated by reversed-phase HPLC; the fractions collected are identified by mass spectrometry. The disulfide structures of the intermediates are then determined by specific chemical cleavage and mass-mapping by MALDI-MS, a novel approach developed in our laboratory. The procedure of quenching and trapping of disulfide intermediates in acidic solution minimizes sulfhydryl-disulfide exchange, and therefore provides a good measure of folding kinetics and preservation of intermediate species. Our cyanylation methodology for disulfide mapping is simpler, faster, and more sensitive than the more conventional approach. Among 18 folding intermediates isolated and identified at different time points, disulfide structures of seven well-populated intermediates, including two non-native isomers with scrambled disulfide structures, one 2-disulfide intermediate, and four 1-disulfide intermediates, have been characterized; most of them possess non-native disulfide structures.

Nonnative isomers of proline-93 and -114 predominate in heat-unfolded ribonuclease A

The peptide bonds preceding both Pro-93 and Pro-114, which are in the cis conformation in native RNase A, are predominantly in the trans conformation in the heat-unfolded protein. The percentages are estimated to be 60% and 63%, respectively, with a standard deviation of +/- 7% in each quantity. These ratios are close to those found for corresponding sequences in X-Pro-Y peptides. The concentration of the trans proline species was determined from the integrated intensities of resonance peaks of the C alpha H protons of Tyr-92 and Asn-113, which are well resolved in the 1D proton NMR spectrum of heat-unfolded RNase A. The assignments of the resonances were deduced from 2D NOESY and DQF-COSY spectra of unfolded RNase A in D2O. Furthermore, the C alpha H protons of both Tyr-92 and Asn-113 had an intense NOE cross-peak with the C delta H and C delta' H of the respective following prolines. For both Pro-93 and Pro-114, these NOE cross-peaks would arise only if the X-Pro peptide bond were in the trans conformation. It is generally believed that the rate of refolding of RNase A is considerably reduced by nonnative proline isomers, such as trans Pro-93. Two models for folding RNase A, that are consistent with these new results and the work of previous investigators, are presented here.

Refolding of denatured ribonuclease observed by size exclusion chromatography.

The unfolding and refolding of pancreatic ribonuclease have been observed by absorbance, fluorescence, and size exclusion chromatographic measurements in solutions of guanidinium chloride continuously maintained at pH 6.0 and 4 degrees C. The spectral measurements were fitted with a minimal number of kinetic phases while the chromatographic measurements were simulated from an explicit mechanism. All of the measurements are consistent with a minimal mechanism involving seven components. The folded components include the native protein and two transiently stable intermediates each having the same hydrodynamic volume. The intermediate having all native peptide isomers has an unfolding midpoint in 3.8 M denaturant while the intermediate having one nonnative peptide isomer has an unfolding midpoint in 1.3 M denaturant. The unfolded protein is distributed among four components having the same hydrodynamic volume but differing peptide isomers. At equilibrium, 10% of the denatured protein has all native isomers, 60% has one nonnative isomer, 5% has a different nonnative isomer, and 25% has both nonnative isomers. In low denaturant concentrations, the dominant component with one nonnative isomer can refold to transiently populate the compact intermediate with the same nonnative isomer.

Catalysis of protein folding by prolyl isomerase

Rates of protein folding reactions vary considerably. Some denatured proteins regain the native conformation within milliseconds or seconds, whereas others refold very slowly in the time range of minutes or hours. Varying folding rates are observed not only for different proteins, but can also be detected for single polypeptide species. This originates from the co-existence of fast- and slow-folding forms of the unfolded protein, which regain the native state with different rates. The proline hypothesis provides a plausible explanation for this heterogeneity. It assumes that the slow-folding molecules possess non-native isomers of peptide bonds between proline and another residue, and that crucial steps in the refolding of the slow-folding molecules are limited in rate by the slow reisomerization of such incorrect proline peptide bonds. Recently the enzyme peptidyl-prolyl cis-trans isomerase (PPIase) was discovered and purified from pig kidney. It catalyses efficiently the cis in equilibrium trans isomerization of proline imidic peptide bonds in oligopeptides. Here we show that it also catalyses slow steps in the refolding of a number of proteins of which fast- and slow-folding species have been observed and where it was suggested that proline isomerization was involved in slow refolding. The efficiency of catalysis depends on the accessibility for the isomerase of the particular proline peptide bonds in the refolding protein chain.

Effects of guanidine hydrochloride on the refolding kinetics of denatured thioredoxin

The effect of guanidine hydrochloride concentration on the kinetics of the conformational change of Escherichia coli thioredoxin was examined by using fluorescence, absorbance, circular dichroic, and viscosity measurements. Native thioredoxin unfolds in a single kinetic phase whose time constant decreases markedly with increasing denaturant concentration in the denaturation base-line zone. This dependency merges with the time constant of the slowest refolding kinetic phase at the midpoint of the equilibrium transition in 2.5 M denaturant. The time constant of the slowest refolding phase becomes denaturant independent below 1 M denaturant in the native base-line region. The denaturant-independent slowest refolding phase has an activation energy of 16 kcal/mol and is generated in the denatured base-line zone in a denaturant-independent reaction having a time constant of 19 s at 25 degrees C. The fractional amplitude of the slowest refolding phase diminishes in the native base-line zone to a minimum value of 0.25. This decrease is accompanied by an increase in the fractional amplitudes of two faster refolding kinetic phases, an increase describing a sigmoidal transition centered at about 1.6 M denaturant. Manual multimixing measurements indicate that only the slowest refolding kinetic phase generates a product having the stability of the native protein. We suggest that the two faster refolding phases reflect the transient accumulation of folding intermediates which can contain a nonnative isomer of proline peptide 76.

On the renaturation of reduced hen egg white lysozyme containing two blocked sulfhydryl groups

Formation of native lysozyme from the reduced form involves many pathways in two processes: incorrect pairing of half-cystine residues by oxidation and rearrangement of disulfide (SS) bonds. The energy barrier against suflhydryl (SH)-disulfide interchange of the native or nativelike species thus formed causes accumulation of these species. For example, the enzymatically active isomers containing three presumably native SS bonds and one open SS bond may be thermodynamically favorable over the nonnative isomers and can be formed from reduced lysozyme or lysozyme containing scrambled SS bonds by nonobligatory and flexible pathways. As an extension of these observations formation of nativelike species from reduced lysozyme containing the average of two carboxymethyl (CM)-cysteine was investigated.