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• W2109131504 abstract "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. 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. Although it is widely accepted that the folding of proteins is governed exclusively by their amino acid sequence (1Anfinsen C.B. Science. 1973; 181: 223-230Crossref PubMed Scopus (5107) Google Scholar), the prediction of the three-dimensional structure of a biologically active protein from its primary sequence remains an unsolved challenge. The importance of protein folding is highlighted by the causes of debilitating diseases such as Alzheimer disease, cystic fibrosis, and Creutzfeld-Jakob disease (2Dobson C.M. Semin. Cell Dev. Biol. 2004; 15: 3-16Crossref PubMed Scopus (714) Google Scholar, 3Stefani M. Biochim. Biophys. Acta. 2004; 1739: 5-25Crossref PubMed Scopus (367) Google Scholar), which are attributed to the loss of biological functions of specific proteins due to their inability to either fold or remain correctly folded. An understanding of protein folding should provide a greater opportunity to devise novel approaches for the treatment of these and other protein misfolding diseases. In proteins containing cysteine residues, the oxidative formation of native disulfide bonds is an integral part of the folding process. In such proteins, conformational folding is coupled with disulfide formation in the process of oxidative folding. Because disulfide bonds play key roles in the stabilization of three-dimensional structures in vivo but their formation in a cellular environment is poorly understood, the study of oxidative folding in vitro offers a valuable tool to understand the complex pathways by which native disulfide formation takes place. In particular, the study of oxidative folding is facilitated by the ability to isolate discrete (disulfide-bonded) intermediates, something that cannot be achieved in studies of conformational folding of non-disulfide-containing proteins. Model studies of oxidative folding of cysteine-rich proteins, such as bovine pancreatic trypsin inhibitor (4Creighton T. J. Mol. Biol. 1974; 87: 579-602Crossref PubMed Scopus (84) Google Scholar, 5Weissman J.S. Kim P.S. Science. 1991; 253: 1386-1393Crossref PubMed Scopus (487) Google Scholar) and ribonuclease A (6Wedemeyer W.J. Welker E. Narayan M. Scheraga H.A. Biochemistry. 2000; 39: 4207-4216Crossref PubMed Scopus (514) Google Scholar, 7Wedemeyer W.J. Xu X. Welker E. Scheraga H.A. Biochemistry. 2002; 41: 1483-1491Crossref PubMed Scopus (22) Google Scholar) have established a basis for understanding this process. Most oxidative folding studies reported to date have involved the isolation of stable intermediates by liquid chromatographic purification of acid-quenched folding reactions followed by structural elucidation and disulfide connectivity analysis. The oxidative folding pathways analyzed so far can be classified in terms of the number and types (i.e. native or non-native) of disulfide bonds present in the intermediate species. Interestingly, different combinations of intermediates have been found for different proteins, and it is not clear how the primary amino acid sequence or a particular three-dimensional fold relates to a particular type of oxidative folding pathway. Thus there is a need for detailed studies on specific protein classes, and in this paper, we report on a class where the final disulfide network is particularly interesting in that it forms a “knotted” structure. Specifically, the current study is directed at understanding the oxidative folding process for an intriguing class of cystine knot proteins that also have a macrocyclic backbone (8Mulvenna J.P. Wang C. Craik D.J. Nucleic Acids Res. 2006; 34: D192-D194Crossref PubMed Scopus (131) Google Scholar, 9Trabi M. Craik D.J. Trends Biochem. Sci. 2002; 27: 132-138Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). In cystine knot proteins (10Pallaghy P.K. Nielsen K.J. Craik D.J. Norton R.S. Protein Sci. 1994; 3: 1833-1839Crossref PubMed Scopus (463) Google Scholar, 11Craik D.J. Daly N.L. Waine C. Toxicon. 2001; 39: 43-60Crossref PubMed Scopus (409) Google Scholar), two disulfide bonds and their connecting backbone segments form an embedded ring that is penetrated by a third disulfide bond, as highlighted in Fig. 1. Oxidative folding studies of cystine knots, such as hirudin (12Chang J.Y. Li L. Bulychev A. J. Biol. Chem. 2000; 275: 8287-8289Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), tick anti-coagulant protein (12Chang J.Y. Li L. Bulychev A. J. Biol. Chem. 2000; 275: 8287-8289Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), Amaranthus α-amylase inhibitor (13Cemazar M. Zahariev S. Lopez J.J. Carugo O. Jones J.A. Hore P.J. Pongor S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5754-5759Crossref PubMed Scopus (55) Google Scholar, 14Cemazar M. Zahariev S. Pongor S. Hore P.J. J. Biol. Chem. 2004; 279: 16697-16705Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), and potato carboxypeptidase inhibitor (15Chang J.Y. Li L. Canals F. Aviles F.X. J. Biol. Chem. 2000; 275: 14205-14211Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) reported a highly heterogeneous mix of one-, two-, and three-disulfide intermediates, among which some contained non-native disulfide bonds. Another cystine knot, the trypsin-specific inhibitor from the squash plant Ecballium elaterium (EETI-II) 4The abbreviations used are: EETI-II, E. elaterium trypsin inhibitor I; MCoTI-II, M. cochinchinensis trypsin inhibitor II; CCK, cyclic cystine knot; CM, carboxyamidomethylated; RP-HPLC, reverse-phase high performance liquid chromatography; DTT, dithiothreitol; MS-MS, tandem mass spectrometry; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; ppm, parts/million. 4The abbreviations used are: EETI-II, E. elaterium trypsin inhibitor I; MCoTI-II, M. cochinchinensis trypsin inhibitor II; CCK, cyclic cystine knot; CM, carboxyamidomethylated; RP-HPLC, reverse-phase high performance liquid chromatography; DTT, dithiothreitol; MS-MS, tandem mass spectrometry; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; ppm, parts/million. (16Le-Nguyen D. Heitz A. Chiche L. el Hajji M. Castro B. Protein Sci. 1993; 2: 165-174Crossref PubMed Scopus (64) Google Scholar) forms a predominant oxidative folding intermediate that is only partially oxidized and contains only native disulfide bonds. This kind of folding pathway has also been detected for other cysteine-rich proteins that are not cystine knots, for example epidermal growth factor (17Chang J.Y. Li L. Lai P.H. J. Biol. Chem. 2001; 276: 4845-4852Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), hen egg white lysozyme (18van den Berg B. Chung E.W. Robinson C.V. Dobson C.M. J. Mol. Biol. 1999; 290: 781-796Crossref PubMed Scopus (80) Google Scholar), and insulin-like growth factor I (19Miller J.A. Narhi L.O. Hua Q.X. Rosenfeld R. Arakawa T. Rohde M. Prestrelski S. Lauren S. Stoney K.S. Tsai L. Weiss M.A. Biochemistry. 1993; 32: 5203-5213Crossref PubMed Scopus (103) Google Scholar). The class of proteins we have examined here is the cyclotides (20Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (635) Google Scholar, 21Craik D.J. Daly N.L. Mulvenna J. Plan M.R. Trabi M. Curr. Protein Pept. Sci. 2004; 5: 297-315Crossref PubMed Scopus (161) Google Scholar), which are characterized by a cystine knot that is embedded within a macrocyclic backbone, defining a motif referred to as the cyclic cystine knot (CCK). As might be imagined, the combination of a macrocyclic backbone and a knotted cross-bracing motif makes the cyclotides extremely stable, and they maintain structure and biological activity after extremes of thermal, proteolytic and chemical exposure (22Colgrave M.L. Craik D.J. Biochemistry. 2004; 43: 5965-5975Crossref PubMed Scopus (448) Google Scholar, 23Gran L. Sandberg F. Sletten K. J. Ethnopharmacol. 2000; 70: 197-203Crossref PubMed Scopus (122) Google Scholar). As well as being exceptionally stable, the cyclotides are functionally diverse and display a wide range of activities, including antimicrobial activity, enzyme inhibition, and anti-HIV activity as well as insecticidal properties, making them attractive candidates for both drug design and agricultural applications, both in their native forms and as molecular scaffolds for the incorporation of novel bioactivities (24Clark R.J. Daly N.L. Craik D.J. Biochem. J. 2006; 34: 85-93Crossref Scopus (136) Google Scholar, 25Craik D. Cemazar M. Daly N.L. Curr. Opin. Drug Discovery Dev. 2006; (in press)PubMed Google Scholar). Given the topological complexity of the CCK motif, it is of great interest to determine how it is formed; i.e. what is the order in which the three interlocked disulfide bonds are made? A preliminary study on the prototypic cyclotide kalata B1 showed that this uterotonic peptide folds to its native disulfide connectivity via a pathway that involves accumulation of a two-disulfide native-like intermediate species that surprisingly is not a direct precursor of the native species (26Craik D.J. Daly N.L. Protein Pept. Lett. 2005; 12: 147-152Crossref PubMed Scopus (19) Google Scholar, 27Daly N.L. Clark R.J. Craik D.J. J. Biol. Chem. 2003; 278: 6314-6322Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 28Daly N.L. Clark R.J. Goransson U. Craik D.J. Lett. Pept. Sci. 2003; 10: 523-531Crossref Scopus (12) Google Scholar). Given this surprising result, it was of interest to examine oxidative folding in another subclass of CCK protein. In the current study, we examine the oxidative folding of MCoTI-II (Momordica cochinchinensis trypsin inhibitor II). As the name suggests, this molecule is a trypsin inhibitor isolated from seeds of the tropical fruit M. cochinchinensis. It was first isolated in 2000 (29Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. Andrieu J.P. Heitz A. Trinh Hong T. Pham T.T. Le Nguyen D. Biochemistry. 2000; 39: 5722-5730Crossref PubMed Scopus (293) Google Scholar), and its structure was independently determined soon after by two groups (30Felizmenio-Quimio M.E. Daly N.L. Craik D.J. J. Biol. Chem. 2001; 276: 22875-22882Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 31Heitz A. Hernandez J.F. Gagnon J. Hong T.T. Pham T.T. Nguyen T.M. Le-Nguyen D. Chiche L. Biochemistry. 2001; 40: 7973-7983Crossref PubMed Scopus (148) Google Scholar). Fig. 1 shows the sequence and structure of MCoTI-II and highlights the disulfide bonds arranged in a CCK motif. MCoTI-II belongs to the trypsin inhibitory subfamily of cyclotides and is quite different in sequence to the other two subfamilies, the Möbius subfamily of which kalata B1 is a member, and the bracelet subfamily (28Daly N.L. Clark R.J. Goransson U. Craik D.J. Lett. Pept. Sci. 2003; 10: 523-531Crossref Scopus (12) Google Scholar, 29Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. Andrieu J.P. Heitz A. Trinh Hong T. Pham T.T. Le Nguyen D. Biochemistry. 2000; 39: 5722-5730Crossref PubMed Scopus (293) Google Scholar). Indeed, it is more homologous to a family of acyclic trypsin inhibitors from plants in the Cucurbitaceae family known as squash protease inhibitors. These include EETI-II (32Kratzner R. Debreczeni J.E. Pape T. Schneider T.R. Wentzel A. Kolmar H. Sheldrick G.M. Uson I. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 1255-1262Crossref PubMed Scopus (37) Google Scholar, 33Heitz A. Chiche L. Le-Nguyen D. Castro B. Eur. J. Biochem. 1995; 233: 837-846Crossref PubMed Scopus (24) Google Scholar) and Cucurbita maxima trypsin inhibitor-1 (34Thaimattam R. Tykarska E. Bierzynski A. Sheldrick G.M. Jaskolski M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1448-1461Crossref PubMed Scopus (20) Google Scholar, 35Helland R. Berglund G.I. Otlewski J. Apostoluk W. Andersen O.A. Willassen N.P. Smalas A.O. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 139-148Crossref PubMed Scopus (37) Google Scholar) for which structures have been reported both free in solution and bound to the proteases they inhibit. The structure of MCoTI-II is similar to its linear counterparts apart from the presence of a linker joining what would otherwise have been the termini of the protein and forming loop 6 of the macrocyclic backbone (see Fig. 1 for a definition of the loops in cyclotides). The cystine knot is present both in the linear and the cyclic versions of these trypsin inhibitors and is a major stabilizing factor of them. The oxidative folding of EETI-II is characterized by the presence of a native-like two-disulfide intermediate species that is a direct precursor of the native species (33Heitz A. Chiche L. Le-Nguyen D. Castro B. Eur. J. Biochem. 1995; 233: 837-846Crossref PubMed Scopus (24) Google Scholar). In the present study, the availability of functionally related cyclic and linear counterparts of a cystine-knotted protein provides an opportunity to examine the contribution of the cyclic backbone to the oxidative folding process of cystine knots. Isolation of MCoTI-II—MCoTI-II was extracted from the dormant seeds of M. cochinchinensis as described previously (29Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. Andrieu J.P. Heitz A. Trinh Hong T. Pham T.T. Le Nguyen D. Biochemistry. 2000; 39: 5722-5730Crossref PubMed Scopus (293) Google Scholar) and purified using RP-HPLC. Masses were analyzed using an electrospray ionization time-of-flight Micromass LCT mass spectrometer. Oxidative Folding and Reductive Unfolding—Oxidative folding was performed in 0.1 m NH4OAc, pH 8.5, 0.1 mg/ml MCoTI-II at 25 °C, containing varying concentrations of reduced glutathione from 1 to 5 mm to mimic the biochemical environment of oxidative folding in vivo. For reductive unfolding, purified native or IIa species (0.1 mg/ml) was dissolved in 0.1 m NH4HCO3, pH 8.0, containing 75 mm DTT at 25 °C. Aliquots were removed at various time intervals and quenched with 4% trifluoroacetic acid. Samples were analyzed using a Phenomenex RP-HPLC C18 column (150 × 4.6 mm, 3 μm) with linear gradients of solvents A (H2O/0.05% trifluoroacetic acid) and B (90% CH3CN/10% H2O/0.05% trifluoroacetic acid) at a flow rate of 0.3 ml/min. Carboxyamidomethylation—Purified IIa was dissolved in 0.5 m Tris acetate, pH 8.0, containing 2 mm Na2EDTA and an excess of iodoacetamide. The reaction was quenched after 1 min with 0.5 m sodium citrate, pH 3.0. The reaction mixture was purified on a semipreparative RP-HPLC (250 × 10 mm, 4 μm) C18 column. Enzymatic Digestion and Nanospray MS-MS Sequencing—The disulfide connectivities of intermediate IIa were determined via trypsin and/or α-chymotrypsin digestion of the reduced carboxyamidomethylated analogue of IIa. After purification to >98%, this species was incubated at 37 °C in 0.1 m NH4HCO3, pH 8.0, with a protease:protein ratio of 1:20. The digestions were quenched after 2 h by the addition of an equal volume of 0.5% formic acid and desalted using Ziptips (Millipore). The fragments resulting from the digestion were examined first by matrix-assisted laser desorption ionization time-of-flight mass spectrometry followed by sequencing by nanospray MS-MS on a QStar mass spectrometer. A capillary voltage of 900 V was applied, and spectra were acquired between m/z 60-2000 for both time-of-flight spectra and product ion spectra. The collision energy for peptide fragmentation was varied between 10 and 50 V, depending on the size and charge of the ion. MS-MS spectra were examined and sequenced based on the presence of both b and y series of ions present (N- and C-terminal fragments). NMR Experiments—Samples for NMR spectroscopy were prepared by dissolving the protein in 90% H2O, 10% D2O, and 0.1% trifluoroacetic acid to final concentrations of 0.5-0.7 mm. The trifluoroacetic acid was used to maintain a constant pH of 2 to prevent the oxidation and/or reshuffling of cysteine residues. Spectra were recorded on Bruker ARX 500 or Bruker ARX 600 NMR spectrometers at a temperature of 290 K. For TOCSY experiments, the mixing time was 80 ms and for NOESY, the mixing time was 200 ms. Slowly exchanging amide protons were identified by recording a series of one-dimensional and TOCSY spectra at 290 K over 20 h immediately after dissolution of a sample in 100% D2O and 0.1% trifluoroacetic acid. Two-dimensional spectra were collected with 4096 data points in the f2 dimension and 512 increments in the f1 and processed using Topspin (Bruker) software. The f1 dimension was zero-filled to 2048 real data points, with the f1 and f2 dimensions multiplied by a sine-squared function prior to Fourier transformation. One-dimensional spectra were recorded with 32,768 points over a 12-ppm spectral width. The main aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic knotted peptide MCoTI-II. Put simply, how do 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 knotted topology? To answer this question, which is summarized in Fig. 1, we isolated a sample of the naturally occurring peptide and studied its folding/unfolding using a range of spectroscopic methods. The native peptide was extracted from the seeds of M. cochinchinensis as described previously (29Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. Andrieu J.P. Heitz A. Trinh Hong T. Pham T.T. Le Nguyen D. Biochemistry. 2000; 39: 5722-5730Crossref PubMed Scopus (293) Google Scholar) and purified over several rounds of preparative and semipreparative RP-HPLC. The identity and purity of the isolated peptide were verified by mass spectrometry. Oxidative Folding and Reductive Unfolding of MCoTI-II—The oxidative folding pathway of MCoTI-II was examined at the level of individual disulfide species using acid quench RP-HPLC. First, oxidative folding of cyclic reduced species was studied in the presence of different concentrations of reduced glutathione to determine its role in the mechanism and rate of the folding process. Fig. 2 shows RP-HPLC traces that monitor the oxidative folding of the reduced peptide at pH 8.5 in 0.1 m ammonium acetate buffer with 2 mm reduced glutathione. The chromatograms show that the oxidative folding pathway is homogeneous, and there are only two species that are detected in significant amounts, namely the native species (N) and a folding intermediate (IIa). The native species accumulates to >90% of the overall protein content, indicating highly efficient disulfide formation. We also examined the oxidative folding at a higher concentration of reduced glutathione (5 mm) and found that the mechanism did not change by increasing the reducing potential of the folding buffer. Increasing the reducing potential does increase the rate of formation of the native species but not the overall yield. Previously, it has been shown that MCoTI-II is prone to α/β-aspartyl isomerization, which introduces an extra CH2 group into the peptide backbone at the Asp31-Gly32 linkage in loop 6. The isomerization reaction is accelerated by alkaline pH, and the β-aspartyl isomer of MCoTI-II is separately detectable, eluting before the α-isomer on RP-HPLC. Fig. 2A shows that, during refolding of the α-aspartyl isomer, small amounts of the β-aspartyl isomer are obtained, whereas in Fig. 2B, the complementary reaction is evident; from purified reduced β-aspartyl isomer, small amounts of the oxidized α-aspartyl are obtained. The two isomers have been separated on RP-HPLC, and their structures have been studied via two-dimensional NMR (30Felizmenio-Quimio M.E. Daly N.L. Craik D.J. J. Biol. Chem. 2001; 276: 22875-22882Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). We found here that the β-aspartyl isomer of MCoTI-II follows the same oxidative folding mechanism as the α-aspartyl isomer and has a similar rate constant. This shows that small structural perturbations in loop 6 are not crucial for the folding pathway. The reductive unfolding of MCoTI-II was found to be pH-dependent. MCoTI-II was highly resistant to reduction at pH 3 with 20 mm tris(2-carboxyethyl) phosphine hydrochloride and did not produce a significant amount of any of the refolding intermediates or the reduced peptide over 8 h. However, at pH 8.0, reduction with DTT easily produced >90% of fully reduced species within 30 min (Fig. 2C). Similar to the process of oxidative folding, the reductive unfolding pathway features the native (N), fully reduced (R), and the intermediate species (IIa) detected previously and a small number of other intermediate species. It is therefore clear that MCoTI-II is not only oxidized via this intermediate species but is also reduced via this species. The intermediate species was confirmed to be the same in both the oxidative and the reductive process by RP-HPLC retention time, electrospray ionization mass spectrometry, and later by its disulfide content. It was confirmed to be a two-disulfide species by liquid chromatrography-mass spectrometry. Similarly, two other minor intermediates, IIb and IIc, appearing on the reductive unfolding pathway (see Fig. 2C) were found to be two-disulfide species. A mass spectrometric confirmation of the disulfide content of the various species detected in oxidative folding and reductive unfolding is given in Table 1.TABLE 1ESI-MS data for the different disulfide species of MCoTI-IISpeciesTheoretical massExperimental massDaNative (N)3453.53452.8Reduced (R)3459.53458.82 Disulfide intermediates IIa, IIb, and IIc3455.53454.82CM-IIa3569.53568.8 Open table in a new tab Disulfide Bond Connectivity of IIa—It was important to determine the disulfide connectivity of the main two-disulfide intermediate to understand the detailed mechanism of disulfide formation in MCoTI-II. A sample of IIa was isolated by partially reducing native MCoTI-II for 4 min (Fig. 3A), as it was seen in the reductive unfolding studies that, at this time point, the protein exists primarily as the IIa species. The intermediate was purified by RP-HPLC (Fig. 3B) and subjected to carboxyamidomethylation with iodoacetamide. The carboxyamidomethylated species, referred to as 2CM-IIa, was isolated using RP-HPLC with >99% purity (Fig. 3C). Electrospray ionization mass spectrometry of purified IIa and 2CM-IIa confirmed that IIa is a two-disulfide intermediate (Table 1). Hence, it remained to determine the identity of the missing disulfide bond. This was done by fully reducing 2CM-IIa to a species with four thiol groups (Fig. 3D) and then partially digesting it with trypsin and/or α-chymotrypsin for 2 h before sequencing the fragments using tandem mass spectrometry. It was confirmed that IIa is missing the Cys1-Cys18 bond (Table 2).TABLE 2Peptide sequencing of a partial trypsin digest of 2CM 4SH IIaFragment sequenceTheoretical mass, MWTExperimental mass, MWEMWE-MWTCM cysteines (from MS/MS)DaDaDaCys14-Lys32255.862373.87118.012 (Cys1, Cys14)Asp11-Arg211136.401195.4259.021 (Cys14)Asp11-Lys32572.952690.98118.032 (Cys1, Cys14)Arg10-Lys32729.052847.08118.032 (Cys1, Cys14)Cys8-Lys32987.153106.16119.032 (Cys1, Cys14) Open table in a new tab Three-dimensional Structural Features of IIa—After isolating IIa in high purity, it was subjected to two-dimensional NMR analysis to compare its structural features to the native species. Two-dimensional TOCSY and NOESY spectra were obtained for pure native (N), intermediate (IIa), and its derivative species with the two carboxyamidomethylated cysteines (2CM-IIa). The spectra were assigned, and the fingerprint regions for the three species are shown in Fig. 4. The amide signals are well dispersed, and from the large number of cross-peaks in the NOESY spectra, it is clear that all three species have well defined structures. The chemical shifts of the Hα protons of the native species are the same as those reported previously (30Felizmenio-Quimio M.E. Daly N.L. Craik D.J. J. Biol. Chem. 2001; 276: 22875-22882Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). A plot of Hα chemical shifts against the residue number for the native, IIa, and 2CM-IIa species shows that IIa and 2CM-IIa are structurally very similar to the native species (Fig. 5A), with the shift differences rarely exceeding 0.1 ppm for any given Hα in the sequence. The main differences in shifts between the species are clustered near Cys1-Ile4 and Ala17-Ile19. Figs. 5, B and C, highlight the regions in IIa and 2CM-IIa that differ by >0.1 ppm in chemical shift from the native protein and illustrate that the regions coincide exactly with the missing native disulfide bond Cys1-Cys18 and nearby residues. Because the rest of the chemical shifts are similar to those of the native protein, it can be inferred that the two disulfides of IIa are the two remaining native disulfides of the cystine knot (Cys8-Cys20, Cys14-Cys26; i.e. CysII-CysV and CysIII-CysVI; see Fig. 1). Oxidative Folding and Reductive Unfolding of IIa—To determine whether IIa is a direct precursor of native MCoTI-II, the oxidative folding and reductive unfolding of this intermediate were studied. Fig. 6A shows RP-HPLC traces of the oxidative folding process from purified IIa under the same conditions as pure reduced species was folded in Fig. 2A. There are no other species present in significant amounts on this pathway, and IIa directly interconverts to the native species. Furthermore, we found that IIa readily interconverts to the native species even in the absence of oxidative conditions and at low pH. After two-dimensional NMR studies were performed, it became clear that IIa does not have prolonged stability even at pH 2, as after 52 h in the NMR tube, 25% had interconverted to native MCoTI-II (data not shown). This finding has double significance. First, IIa under these conditions is stable enough to perform the two-dimensional NMR experiments needed for spectral assignment (Fig. 4), which take ∼24 h. Second, it is clear that, even at low pH where the rate of disulfide formation is usually very low, the native species is readily formed from IIa, and no other species were present when this sample was analyzed using RP-HPLC. This finding points to the fact that IIa converts directly into the native species. Reductive unfolding from purified IIa features some other minor intermediates that have been previously seen on the reductive unfolding pathway of native MCoTI-II (Fig. 6B). Overall, IIa has a similar mechanism of reductive unfolding as the native species. Thermal Stability of the Cystine Knot in MCoTI-II—One-dimensional NMR spectra of native MCoTI-II and 2CM-IIa were recorded at a range of temperatures to compare the thermal stability of the peptides. Solutions of both peptides were heated from 293 to 353 K and cooled" @default.
• W2109131504 created "2016-06-24" @default.
• W2109131504 creator A5009476285 @default.
• W2109131504 creator A5020747744 @default.
• W2109131504 creator A5079308758 @default.
• W2109131504 creator A5080112340 @default.
• W2109131504 creator A5080238392 @default.
• W2109131504 creator A5087586811 @default.
• W2109131504 date "2006-03-01" @default.
• W2109131504 modified "2023-10-13" @default.
• W2109131504 title "Knots in Rings" @default.
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