Knots in rings. The circular knotted protein Momordica cochinchinensis trypsin inhibitor-II folds via a stable two-disulfide intermediate

The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys1-Cys18 disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability.     

Pathway of oxidative folding of alpha-lactalbumin: a model for illustrating the diversity of disulfide folding pathways

The pathway of oxidative folding of alpha-lactalbumin (alpha LA) (four disulfide bonds) has been characterized by structural and kinetic analysis of the acid-trapped folding intermediates. In the absence of calcium, oxidative folding of alpha LA proceeds through highly heterogeneous species of one-, two-, three-, and four-disulfide (scrambled) intermediates to reach the native structure. In the presence of calcium, the folding intermediates of alpha LA comprise two predominant isomers (alpha LA-IIA and alpha LA-IIIA) adopting exclusively native disulfide bonds, including the two disulfide bonds (Cys(61)-Cys(77) and Cys(73)-Cys(91)) located within the beta-sheet calcium binding domain. alpha LA-IIA is a two-disulfide species consisting of Cys(61)-Cys(77) and Cys(73)-Cys(91) disulfide bonds. alpha LA-IIIA contains Cys(61)-Cys(77), Cys(73)-Cys(91), and Cys(28)-Cys(111) disulfide bonds. The underlying mechanism of the contrasting folding pathways of calcium-bound and calcium-depleted alpha LA is congruent with the cause of diversity of disulfide folding pathways observed among many well-characterized three-disulfide proteins, including bovine pancreatic trypsin inhibitor and hirudin. Our study also reveals novel aspects of the folding mechanism of alpha LA that have not been described previously.     

Pathway of oxidative folding of bovine alpha-interferon: predominance of native disulfide-bonded folding intermediates

Bovine alpha-interferon (BoINF-alpha) is a single polypeptide protein containing 166 amino acids, two disulfide bonds (Cys1-Cys99 and Cys29-Cys138), and five stretches of alpha-helical structure. The pathway of oxidative folding of BoINF-alpha has been investigated here. Of the eight possible one- and two-disulfide isomers, only two nativelike one-disulfide isomers, BoINF-alpha (Cys1-Cys99) and BoINF-alpha (Cys29-Cys138), predominate as intermediates along the folding pathway. More strikingly, alpha-helical structures formed almost quantitatively before any detectable formation of a disulfide bond. This is demonstrated by the observation that fully reduced BoINF-alpha (starting material of oxidative folding) and reduced carboxymethylated BoINF-alpha both exhibit alpha-helical structure content indistinguishable form that of native BoINF-alpha. The folding mechanism of BoINF-alpha appears to be compatible with the framework model, in which secondary structures fold first, followed by docking (compaction) of preformed secondary structural elements yielding the native structure.     

Disulfide folding pathways of cystine knot proteins. Tying the knot within the circular backbone of the cyclotides

The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (Le-Nguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.     

Energetics of structural domains in alpha-lactalbumin.

alpha-Lactalbumin is a small, globular protein that is stabilized by four disulfide bonds and contains two structural domains. One of these domains is rich in alpha-helix (the alpha-domain) and has Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds. The other domain is rich in beta-sheet (the beta-domain), has Cys 61-Cys 77 and Cys 73-Cys 91 disulfide bonds, and includes one calcium binding site. To investigate the interaction between domains, we studied derivatives of bovine alpha-lactalbumin differing in the number of disulfide bonds, using calorimetry and CD at different temperatures and solvent conditions. The three-disulfide form, having a reduced Cys 6-Cys 120 disulfide bond with carboxymethylated cysteines, is similar to intact alpha-lactalbumin in secondary and tertiary structure as judged by its ellipticity in the near and far UV. the two-disulfide form of alpha-lactalbumin, having reduced Cys 6-Cys 120 and Cys 28-Cys 111 disulfide bonds with carboxymethylated cysteines, retains about half the secondary and tertiary structure of the intact alpha-lactalbumin. The remaining structure is able to bind calcium and unfolds cooperatively upon heating, although at lower temperature and with significantly lower enthalpy and entropy. We conclude that, in the two disulfide form, alpha-lactalbumin retains its calcium-binding beta-domain, whereas the alpha-domain is unfolded. It appears that the beta-domain does not require alpha-domain to fold, but its structure is stabilized significantly by the presence of the adjacent folded alpha-domain.     

Local interactions and the role of the 6-120 disulfide bond in alpha-lactalbumin: implications for formation of the molten globule state

Molten globule states are partially folded states of proteins which are compact and contain a high degree of secondary structure but which lack many of the fixed tertiary interactions associated with the native state. A set of peptides has been prepared in order to probe the role of local interactions in the vicinity of the Cys(6)-Cys(120) disulfide bond in stabilizing the molten globule state of human alpha-lactalbumin. Peptides derived from the N-terminal and C-terminal regions of human alpha-lactalbumin have been analyzed using nuclear magnetic resonance, circular dichroism, fluorescence spectroscopy and sedimentation equilibrium experiments. A peptide corresponding to the first helical region in the native protein, residues 1-13, is only slightly helical in isolation. Extending the peptide to include residues 14-18 results in a modest increase in helicity. A peptide derived from the C-terminal 12 residues, residues 112-123, is predominantly unstructured. Crosslinking the N- and C-terminal peptides by the native disulfide bond results in almost no increase in structure and there is no evidence for any significant cooperative structure formation over the range of pH 2.2-11.7. These results demonstrate that there is very little enhancement of local structure due to the formation of the Cys(6)-Cys(120) disulfide bond. This is in striking contrast to peptides derived from the region of the Cys(28)-Cys(111) disulfide.     

Solution structure and activity of the synthetic four-disulfide bond Mediterranean mussel defensin (MGD-1)

MGD-1 is a 39-residue defensin-like peptide isolated from the edible Mediterranean mussel, Mytilus galloprovincialis. This peptide is characterized by the presence of four disulfide bonds. We report here its solid-phase synthesis and an easy way to improve the yield of the four native disulfide bonds. Synthetic and native MGD-1 display similar antibacterial activity, suggesting that the hydroxylation of Trp28 observed in native MGD-1 is not involved in the antimicrobial effect. The three-dimensional solution structure of MGD-1 has been established using (1)H NMR and mainly consists of a helical part (Asn7-Ser16) and two antiparallel beta-strands (Arg20-Cys25 and Cys33-Arg37), together giving rise to the common cystine-stabilized alpha-beta motif frequently observed in scorpion toxins. In MGD-1, the cystine-stabilized alpha-beta motif is stabilized by four disulfide bonds (Cys4-Cys25, Cys10-Cys33, Cys14-Cys35, and Cys21-Cys38), instead of by the three disulfide bonds commonly found in arthropod defensins. Except for the Cys21-Cys38 disulfide bond which is solvent-exposed, the three others belong to the particularly hydrophobic core of the highly constrained structure. Moreover, the C4-P5 amide bond in the cis conformation characterizes the MGD-1 structure. MGD-1 and insect defensin A possess similar bactericidal anti-Gram-positive activity, suggesting that the fourth disulfide bond of MGD-1 is not essential for the biological activity. In agreement with the general features of antibacterial peptides, the MGD-1 and defensin A structures display a typical distribution of positively charged and hydrophobic side chains. The positively charged residues of MGD-1 are located in three clusters. For these two defensin peptides isolated from insects and mollusks, it appears that the rather well conserved location of certain positively charged residues and of the large hydrophobic cluster are enough to generate the bactericidal potency and the Gram-positive specificity.     

Characterization of an alternative low energy fold for bovine α-lactalbumin formed by disulfide bond shuffling

Bovine α-lactalbumin (αLA) forms a misfolded disulfide bond shuffled isomer, X-αLA. This X-αLA isomer contains two native disulfide bridges (Cys 6-Cys 120 and Cys 28-Cys 111) and two non-native disulfide bridges (Cys 61-Cys 73 and Cys 77-Cys 91). MD simulations have been used to characterize the X-αLA isomer and its formation via disulfide bond shuffling and to compare it with the native fold of αLA. In the simulations of the X-αLA isomer the structure of the α-domain of native αLA is largely retained in agreement with experimental data. However, there are significant rearrangements in the β-domain, including the loss of the native β-sheet and calcium binding site. Interestingly, the energies of X-αLA and native αLA in simulations in the absence of calcium are closely similar. Thus, the X-αLA isomer represents a different low energy fold for the protein. Calcium binding to native αLA is shown to help preserve the structure of the β-domain of the protein limiting possibilities for disulfide bond shuffling. Hence, binding calcium plays an important role in both maintaining the native structure of αLA and providing a mechanism for distinguishing between folded and misfolded species.     

Refolding mechanism of ovalbumin: investigation by using a starting urea-denatured disulfide isomer with mispaired CYS367-CYS382

Ovalbumin, a member of the serpin superfamily, contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) in the native state. To investigate the folding mechanism of ovalbumin, a urea-denatured disulfide isomer with a mispaired disulfide Cys367-Cys382 (D[367-382]) and its derivative (D[367-382/CM-73]) in which a native cystine counterpart of Cys73 is blocked by carboxymethylation were produced. Both the denatured isomers refolded within an instrumental dead time of 4 ms into an initial burst intermediate IN with partially folded conformation. After the initial burst phase, most of the D[367-382] molecules further refolded into the native form. In contrast, upon dilution of D[367-382/CM-73] with the refolding buffer, the protein stayed in the IN state as a stable form, which displayed a partial regain of the native secondary structure and a compact conformation with a similar Stokes radius to the native form. The structural characteristics of IN were clearly differentiated from those of an equilibrium intermediate IA that was produced by dilution with an acidic buffer of urea-denatured ovalbumin; IA showed much more hydrophobic dye binding and a larger Stokes radius than the IN state, despite their indistinguishable far-UV circular dichroic spectra. The non-productive nature of IA highlighted the importance of a compact conformation of the IN state for subsequent native refolding. These observations were consistent with a refolding model of ovalbumin that includes the regain of the partial secondary structure and of the compactness of overall conformation in an initial burst phase before the subsequent native refolding.     

A novel endogenous inhibitor of phenoloxidase from Musca domestica has a cystine motif commonly found in snail and spider toxins

Phenoloxidase inhibitor (POI), found in the hemolymph of housefly pupae, is a novel dopa-containing and cystine-rich peptide that competitively inhibits phenoloxidase with a Ki in the nanomolar range. [Tyr32]POI is a potential precursor molecule also found in the hemolymph that may be posttranslationally oxidized to the dopa-containing peptide after creation of a rigid structure. By employing both a solid-phase peptide synthesis system based on a 9-fluorenylmethoxycarbonyl strategy and a specific air oxidation technique to ensure correct folding, we have been able to synthesize [Tyr32]POI. The synthetic [Tyr32]POI was confirmed to be identical to the native [Tyr32]POI by coelution high-performance liquid chromatography analysis and by enzymatic analysis using the phenoloxidase inhibition assay. To determine the disulfide pairings within the peptides, a series of enzyme hydrolyses and partial reduction/alkylation steps were performed. Three cystine pairs (Cys11-Cys25, Cys18-Cys29, and Cys24-Cys36) were determined by identification of the resulting peptides. The disulfide pairings of the two adjacent Cys residues (Cys11-Cys25 and Cys24-Cys36) were unambiguously assigned by comparing the derived fragments with the two possible isomers synthesized through a novel disulfide-linking technique. The arrangement of the disulfide bridges in POI was found to be topologically identical to those found for several peptides within the inhibitor cystine knot structural family. Although these peptides share a low primary sequence homology and display a diversity of biological functions, they nonetheless share similarities in their cystine motifs and tertiary structure. The tertiary structure model of POI, which was derived through molecular dynamics and energy minimization studies using restraints with determined disulfide connectivities, suggests that POI is a new class member of the inhibitor cystine-knot structural family.     

Entropic folding pathway of human epidermal growth factor explored by disulfide scrambling and amplified collective motion simulations

Epidermal growth factor (EGF) regulates cell proliferation and differentiation by binding to the EGF receptor (EGFR) extra-cellular domains. Human EGF is a small, single-chain protein comprising three distinct loops (A, B, and C), which are connected by three disulfide bridges (Cys6-Cys20, Cys14-Cys31, and Cys33-Cys42). These disulfide bridges are essential for structural stability and biological activity. EGF was extensively studied by disulfide scrambling, an experimental technique for the conformational entrapment of intermediate states, which allows us to study the folding pathway of proteins containing disulfide bonds. The experimental results showed that there is a major 2-disulfide intermediate (denoted EGF-II) and that the native disulfide bonding pattern is less prevalent in one of the mutants. In this article, we investigated for the first time the solution conformations of wild-type EGF, EGF-II, and the mutant S9C through extensive molecular dynamics (MD) simulations in water using both the standard MD technique and a recently developed amplified-collective-motion (ACM) sampling method. Compared to standard MD simulations, we achieved a much more enhanced sampling by the ACM simulations, and the structures were sufficiently relaxed to estimate configurational entropies. The simulation results suggest a predominantly entropic folding pathway governed by the disorder of three functional loop regions. Although EGF-II exhibits two native disulfide bonds (Cys14-Cys31 and Cys33- Cys42), its large configurational entropy inhibits a direct transition to the native structure in the folding process. When Ser9 is mutated into Cys, a non-native disulfide bridge Cys9- Cys20 is slightly more favorable than the native Cys6-Cys20 because a less constrained N-terminus affords larger entropy. Isomers that are functionally less active also exhibit a more localized dynamics of the functional loop regions, which may suggest a possible mechanism for the modulation of EGF activity.     

Characterization of a recombinant murine interleukin-6: assignment of disulfide bonds

Murine interleukin 6 (mIL-6) has been synthesized as a fusion protein using a lac operon inducible plasmid in Escherichia coli. The first 8 amino acids are from the N-terminus of bacterial beta-galactosidase and the last 175 amino acids are from residue number 12 to the end of native mIL-6. This fusion protein is equipotent with the native molecule in the hybridoma growth factor assay and has comparable receptor binding characteristics. The two disulfide bridges in mIL-6 have been identified by Staphylococcus aureus V8 protease peptide mapping and Edman degradation of cystine-containing peptides. It has been shown that there are disulfide bonds between Cys46-Cys52 and Cys75-Cys85.     

Effect of an alternative disulfide bond on the structure, stability, and folding of human lysozyme

Human lysozyme has four disulfide bonds, one of which, Cys65-Cys81, is included in a long loop of the beta-domain. A cysteine-scanning mutagenesis in which the position of Cys65 was shifted within a continuous segment from positions 61 to 67, with fixed Cys81, has previously shown that only the mutant W64CC65A, which has a nonnative Cys64-Cys81 disulfide, can be correctly folded and secreted by yeast. Here, using the W64CC65A mutant, we investigated the effects of an alternative disulfide bond on the structure, stability, and folding of human lysozyme using circular dichroism (CD) and fluorescence spectroscopy combined with a stopped-flow technique. Although the mutant is expected to have a different main-chain structure from that of the wild-type protein around the loop region, far- and near-UV CD spectra show that the native state of the mutant has tightly packed side chains and secondary structure similar to that of the wild-type. Guanidine hydrochloride-induced equilibrium unfolding transition of the mutant is reversible, showing high stability and cooperativity of folding. In the kinetic folding reaction, both proteins accumulate a similar burst-phase intermediate having pronounced secondary structure within the dead time of the measurement and fold into the native structure by means of a similar folding mechanism. Both the kinetic refolding and unfolding reactions of the mutant protein are faster than those of the wild-type, but the increase in the unfolding rate is larger than that of the refolding rate. The Gibbs' free-energy diagrams obtained from the kinetic analysis suggest that the structure around the loop region in the beta-domain of human lysozyme is formed after the transition state of folding, and thus, the effect of the alternative disulfide bond on the structure, stability, and folding of human lysozyme appears mainly in the native state.     

Two-State Folding of Lysozyme Versus Multiple-State Folding of α-Lactalbumin Illustrated by the Technique of Disulfide Scrambling

The folding of lysozyme and of alpha-lactalbumin exhibits vastly different kinetics and pathways. Existing evidence indicates that folding intermediates of alphaLA form a well-populated equilibrium molten globule state that is absent in the case of hen lysozyme. We demonstrate here such divergent folding mechanisms of lysozyme and alphaLA using the technique of disulfide scrambling. Two extensively unfolded homologous isomers (beads-form) of lysozyme (Cys6-Cys30, Cys64-Cys76, Cys80-Cys94, Cys115-Cys127) and alphaLA (Cys6-Cys28, Cys61-Cys73, Cys77-Cys91, Cys111-Cys120) were allowed to refold in parallel to form the native protein. Folding kinetics was measured by the recovery of the native structure. Folding intermediates, which illustrate the folding pathway, were trapped by quenching disulfide shuffling and were analyzed by reversed-phase high-pressure liquid chromatography. The results revealed that under identical folding conditions, the folding rate of lysozyme is about 30-fold faster than that of alphaLA. Folding intermediates of lysozyme are far less heterogeneous and sparsely populated than those of alphaLA. Numerous predominant on-pathway and off-pathway intermediates observed along the folding pathway of alphaLA are conspicuously absent in the case of lysozyme. The difference is most striking under fast folding conditions performed in the presence of protein disulfide isomerase. Under these conditions, folding of lysozyme undergoes a near two-state mechanism without accumulation of stable folding intermediates.     

The folding nucleus of the insulin superfamily: a flexible peptide model foreshadows the native state

Oxidative folding of insulin-like growth factor I (IGF-I) and single-chain insulin analogs proceeds via one- and two-disulfide intermediates. A predominant one-disulfide intermediate in each case contains the canonical A20-B19 disulfide bridge (cystines 18-61 in IGF-I and 19-85 in human proinsulin). Here, we describe a disulfide-linked peptide model of this on-pathway intermediate. One peptide fragment (19 amino acids) spans IGF-I residues 7-25 (canonical positions B8-B26 in the insulin superfamily); the other (18 amino acids) spans IGF-I residues 53-70 (positions A12-A21 and D1-D8). Containing only half of the IGF-I sequence, the disulfide-linked polypeptide (designated IGF-p) is not well ordered. Nascent helical elements corresponding to native alpha-helices are nonetheless observed at 4 degrees C. Furthermore, (13)C-edited nuclear Overhauser effects establish transient formation of a native-like partial core; no non-native nuclear Overhauser effects are observed. Together, these observations suggest that early events in the folding of insulin-related polypeptides are nucleated by a native-like molten subdomain containing Cys(A20) and Cys(B19). We propose that nascent interactions within this subdomain orient the A20 and B19 thiolates for disulfide bond formation and stabilize the one-disulfide intermediate once formed. Substitutions in the corresponding region of insulin are associated with inefficient chain combination and impaired biosynthetic expression. The intrinsic conformational propensities of a flexible disulfide-linked peptide thus define a folding nucleus, foreshadowing the structure of the native state.     

Pi7, an orphan peptide from the scorpion Pandinus imperator: a 1H-NMR analysis using a nano-NMR Probe

The three-dimensional solution structure of a novel peptide, Pi7, purified from the venom of the scorpion Pandinus imperator, and for which no specific receptor has been found yet, was determined by two-dimensional homonuclear proton NMR methods from a nanomole amount of compound using a nano-nmr probe. Pandinus imperator peptide 7 does not block voltage-dependent K(+)-channels and does not displace labeled noxiustoxin from rat brain synaptosomal membranes. The toxin has 38 amino acid residues and, similarly to Pi1, is stabilized by four disulfide bridges (Cys6-Cys27, Cys12-Cys32, Cys16-Cys34, and Cys22-Cys37). In addition, the lysine at position 26 crucial for potassium-channel blocking is replaced in Pi7 by an arginine. Tyrosine 34, equivalent to Tyr36 of ChTX is present, but the N-terminal positions 1 and 2 are occupied by two acidic residues Asp and Glu, respectively. The dihedral angles and distance restraints obtained from measured NMR parameters were used in structural calculations in order to determine the conformation of the peptide. The disulfide-bridge topology was established using distance restraints allowing ambiguous partners between S atoms combined with NMR-derived structural information. The structure is organized around a short alpha-helix spanning residues Thr9 to Thr20/Gly21 and a beta-sheet. These two elements of secondary structure are stabilized by two disulfide bridges, Cys12-Cys32 and Cys16-Cys34. The antiparallel beta-sheet is composed of two strands extending from Asn22 to Cys34 with a tight turn at Ile28-Asn29 in contact with the N-terminal fragment Ile4 to Cys6.     

A major kinetic trap for the oxidative folding of human epidermal growth factor

The folding pathway of human epidermal growth factor (EGF) has been characterized by structural and kinetic analysis of the acid-trapped folding intermediates. Oxidative folding of the fully reduced EGF proceeds through 1-disulfide intermediates and accumulates rapidly as a single stable 2-disulfide intermediate (designated as EGF-II), which represents up to more than 85% of the total protein along the folding pathway. Among the five 1-disulfide intermediates that have been structurally characterized, only one is native, and nearly all of them are bridges by neighboring cysteines. Extensive accumulation of EGF-II indicates that it accounts for the major kinetic trap of EGF folding. EGF-II contains two of the three native disulfide bonds of EGF, Cys(14)-Cys(31) and Cys(33)-Cys(42). However, formation of the third native disulfide (Cys(6)-Cys(20)) for EGF-II is slow and does not occur directly. Kinetic analysis reveals that an important route for EGF-II to reach the native structure is via rearrangement pathway through 3-disulfide scrambled isomers. The pathway of EGF-II to attain the native structure differs from that of three major 2-disulfide intermediates of bovine pancreatic trypsin inhibitor (BPTI). The dissimilarities of folding mechanism(s) between EGF, BPTI, and hirudin are discussed in this paper.     

Mass spectrometric analysis of posttranslational modifications of a carrot extracellular glycoprotein

Expression of extracellular dermal glycoprotein (EDGP) is induced by biotic or abiotic stress. The amino acid sequence alignment showed that EDGP shared significant homology with proteins from legumes, tomato, Arabidopsis, wheat, and cotton. These proteins are involved in signal transduction or stress response systems. Most of the Cys residues in these proteins are conserved, suggesting that they share similar tertiary structures. Surface plasmon resonance (SPR) analysis shows that EDGP binds a soybean 4-kDa hormone-like peptide (4-kDa peptide) in vitro and reduction of EDGP decreased significantly the binding activity, implying that posttranslational modifications are important for its function. Therefore, we investigated the posttranslational modifications in EDGP using mass spectrometry. As the result, six disulfide bonds in EDGP were identified: Cys(70)-Cys(158), Cys(84)-Cys(89), Cys(97)-Cys(113), Cys(100)-Cys(108), Cys(201)-Cys(426), and Cys(332)-Cys(378). In addition, the N-terminal glutamine was cyclized into pyroglutamic acid. All four putative glycosylation sites were occupied by N-linked glycans, which have similar masses of m/z 1171. Finally, measuring the mass of the native protein showed that the posttranslational modifications of EDGP (pI 9.5) involved only disulfide bonds, N-terminal modification, and glycosylation.     

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.     

Structures of scrambled disulfide forms of the potato carboxypeptidase inhibitor predicted by molecular dynamics simulations with constraints

The structures of two species of potato carboxypeptidase inhibitor with nonnative disulfide bonds were determined by molecular dynamics simulations in explicit solvent using disulfide bond constraints that have been shown to work for the native species. Ten structures were determined; five for scrambled A (disulfide bonds between Cys8-Cys27, Cys12-Cys18, and Cys24-Cys34) and five for the scrambled C (disulfide bonds Cys8-Cys24, Cys12-Cys18, and Cys27-Cys34). The two scrambled species were both more solvent exposed than the native structure; the scrambled C species was more solvent exposed and less compact than the scrambled A species. Analysis of the loop regions indicates that certain loops in scrambled C are more nativelike than in scrambled A. These factors, combined with the fact that scrambled C has one native disulfide bond, may contribute to the observed faster conversion to the native structure from scrambled C than from scrambled A. Results from the PROCHECK program using the standard parameter database and a database specially constructed for small, disulfide-rich proteins indicate that the 10 scrambled structures have correct stereochemistry. Further, the results show that a characteristic feature of small, disulfide-rich proteins is that they score poorly using the standard PROCHECK parameter database. Proteins 2000;40:482-493.