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• W1984406971 abstract "The unfolding and denaturation curves of leech carboxypeptidase inhibitor (LCI) were elucidated using the technique of disulfide scrambling. In the presence of thiol initiator and denaturant, the native LCI denatures by shuffling its native disulfide bonds and transforms into a mixture of scrambled species. 9 of 104 possible scrambled isomers of LCI, amounting to 90% of total denatured LCI, can be distinguished. The denaturation curve that plots the fraction of native LCI converted into scrambled isomers upon increasing concentrations of denaturant shows that the concentration of guanidine thiocyanate and guanidine hydrochloride required to reach 50% of denaturation is 2.4 and 3.6 m, respectively. In contrast, native LCI is resistant to urea denaturation even at high concentration (8 m). The LCI unfolding pathway was defined based on the evolution of the relative concentration of scrambled isoforms of LCI upon denaturation. Two populations of scrambled species suffer variations along the unfolding pathway. One accumulates as intermediates under strong denaturing conditions and corresponds to open or relaxed structures, among which the beads-form isomer is found. The other population shows an inverse correlation between their relative abundances and the denaturing conditions and should have another kind of non-native structure that is more compact than the unfolded state. The rate constants of unfolding of LCI are low when compared with other disulfide-containing proteins. Overall, the results presented in this study show that LCI, a molecule with potential biotechnological applications, has slow kinetics of unfolding and is highly stable. The unfolding and denaturation curves of leech carboxypeptidase inhibitor (LCI) were elucidated using the technique of disulfide scrambling. In the presence of thiol initiator and denaturant, the native LCI denatures by shuffling its native disulfide bonds and transforms into a mixture of scrambled species. 9 of 104 possible scrambled isomers of LCI, amounting to 90% of total denatured LCI, can be distinguished. The denaturation curve that plots the fraction of native LCI converted into scrambled isomers upon increasing concentrations of denaturant shows that the concentration of guanidine thiocyanate and guanidine hydrochloride required to reach 50% of denaturation is 2.4 and 3.6 m, respectively. In contrast, native LCI is resistant to urea denaturation even at high concentration (8 m). The LCI unfolding pathway was defined based on the evolution of the relative concentration of scrambled isoforms of LCI upon denaturation. Two populations of scrambled species suffer variations along the unfolding pathway. One accumulates as intermediates under strong denaturing conditions and corresponds to open or relaxed structures, among which the beads-form isomer is found. The other population shows an inverse correlation between their relative abundances and the denaturing conditions and should have another kind of non-native structure that is more compact than the unfolded state. The rate constants of unfolding of LCI are low when compared with other disulfide-containing proteins. Overall, the results presented in this study show that LCI, a molecule with potential biotechnological applications, has slow kinetics of unfolding and is highly stable. Leech carboxypeptidase inhibitor (LCI) 1The abbreviations used are: LCIleech carboxypeptidase inhibitorX-LCIscrambled LCIPCIpotato carboxypeptidase inhibitorCTX-IIIcardiotoxin IIITAPtick anticoagulant peptideGdnHClguanidine hydrochlorideGdnSCNguanidine thiocyanateHPLChigh pressure liquid chromatographyMALDI-TOFmatrix-assisted laser desorption/ionization-time of flight mass spectrometry is the first metallocarboxypeptidase inhibitor found in leeches (1Reverter D. Vendrell J. Canals Q. Horstmann J. Avilés F.X. Fritz H. Sommerhoff C.P. J. Biol. Chem. 1998; 273: 32927-32933Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). LCI is a cysteine-rich polypeptide of 66 residues that behaves as a tight binding and competitive inhibitor of different types of pancreatic-like carboxypeptidases (A1, A2, B, and plasma carboxypeptidase B) with equilibrium dissociation constants Ki of 0.1–0.4 × 10−9 m (1Reverter D. Vendrell J. Canals Q. Horstmann J. Avilés F.X. Fritz H. Sommerhoff C.P. J. Biol. Chem. 1998; 273: 32927-32933Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The lowest Ki value of LCI is shown for plasma carboxypeptidase B, an enzyme also known as thrombin activable fibrinolysis inhibitor (TAFI), that proteolytically removes C-terminal residues from fibrin and down-regulates plasminogen activation, leading to an attenuation of fibrinolysis (2Wang W. Nagashima M. Schneider M. Morser J. Nesheim M. J. Biol. Chem. 1998; 273: 27176-27181Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 3Bajzar L. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2511-2518Crossref PubMed Scopus (186) Google Scholar). Assuming that the leech secretes LCI during feeding, this inhibitor may help to maintain blood in the fluid state. The profibrinolytic effect of LCI has recently been demonstrated in an in vitro system, 2S. Salamanca et al., manuscript in preparation. suggesting a potential application to human therapy. In this regard knowledge of the stability and folding behavior of LCI constitutes a basis for the development of variants of the molecule with enhanced activity and/or stability. The recently published three-dimensional structure of LCI shows that it folds in a compact domain consisting of a five-stranded antiparallel β-sheet and a short α-helix (4Reverter D. Fernández-Catalán C. Baumgartner R. Pfänder R. Huber R. Bode W. Vendrell J. Holak T.A. Avilés F.X. Nat. Struct. Biol. 2000; 7: 322-328Crossref PubMed Scopus (67) Google Scholar). One of the main contributions to the stability of native LCI arises from the occurrence of four disulfide bridges between cysteines 10–33, 17–61, 18–42, and 21–57, all of them located within regular secondary structure elements. The unstructured C-terminal tail of LCI interacts with the carboxypeptidase in a substrate-like manner, similar to what was previously described for the potato carboxypeptidase inhibitor (PCI). No sequential homology is observed between PCI and LCI except for the C-terminal tail; however, both proteins show the structural feature of being stabilized by disulfide bridges. leech carboxypeptidase inhibitor scrambled LCI potato carboxypeptidase inhibitor cardiotoxin III tick anticoagulant peptide guanidine hydrochloride guanidine thiocyanate high pressure liquid chromatography matrix-assisted laser desorption/ionization-time of flight mass spectrometry For disulfide-containing proteins, unfolding and refolding generally correlate with reduction and oxidation of the native disulfides (5Haber E. Anfinsen C.B. J. Biol. Chem. 1962; 237: 1839-1844Abstract Full Text PDF PubMed Google Scholar, 6Anfinsen C.B. Science. 1973; 181: 223-230Crossref PubMed Scopus (5217) Google Scholar). Since the intermediates generated in these processes can chemically be trapped and characterized, it is possible to derive in detail the disulfide unfolding and refolding pathways. We have recently described a new methodology to determine stability toward denaturants and to elucidate the unfolding pathway of disulfide-containing proteins (7Chang J.-Y. J. Biol. Chem. 1997; 272: 69-75Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar,8Chang J.-Y. J. Biol. Chem. 1999; 274: 123-128Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). This approach is based on the observation that the presence of trace amounts of a thiol initiator during unfolding by denaturants generates a mixture of scrambled species mostly consisting of non-native disulfides that still maintain the native number of disulfide bonds. This method has been applied to characterize the unfolding pathway of several disulfide-containing proteins, namely tick anticoagulant peptide (TAP) (3 disulfides) (8Chang J.-Y. J. Biol. Chem. 1999; 274: 123-128Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), PCI (3 disulfides) (9Chang J.-Y., Li, L. Canals F. Avilés F.X. J. Biol. Chem. 2000; 275: 14205-14211Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), and insulin-growth factor (IGF-1) (3 disulfides) (10Chang J.-Y. Marki W. Lai P.H. Protein Sci. 1999; 8: 1463-1468Crossref PubMed Scopus (18) Google Scholar). In the present work we describe the unfolding pathway and conformational stability of LCI derived from the characterization of the scrambled species generated in the presence of a thiol initiator. The comparison to other disulfide-containing proteins offers an insight into the role of disulfide bonds in guiding the folding pathway and stabilizing the native fold. Recombinant LCI was obtained by heterologous expression in Escherichia coli following a procedure previously described (1Reverter D. Vendrell J. Canals Q. Horstmann J. Avilés F.X. Fritz H. Sommerhoff C.P. J. Biol. Chem. 1998; 273: 32927-32933Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The recombinant protein that contains a construction-added glycine as the N-terminal residue was purified by ion-exchange chromatography on a TSK DEAE column (Amersham Biosciences) followed by reverse-phase HPLC. The protein was over 99% pure, as judged by HPLC, and its molecular mass was confirmed by MALDI-TOF. Urea, guanidine hydrochloride (GdnHCl), guanidine thiocyanate (GdnSCN), and acetonitrile with purities greater than 99%, were obtained from Merck (Darmstadt, Germany). Native LCI (0.5 mg/ml) was dissolved in Tris-HCl buffer (0.1 m, pH 8.4) containing 0.25 mm2-mercaptoethanol and selected concentrations of denaturants (urea, GdnHCl, GdnSCN, or organic solvent). The reaction was allowed to reach equilibrium and was typically performed at 23 °C for 20 h (8Chang J.-Y. J. Biol. Chem. 1999; 274: 123-128Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). To monitor the kinetics of unfolding, aliquots of the sample treated with selected concentrations of GdnHCl were removed at time intervals, quenched with an equal volume of 4% aqueous trifluoroacetic acid, and directly analyzed by HPLC or kept at 20 °C until analysis. The GdnSCN-denatured samples were further purified by gel-filtration (NAP-5 columns from Amersham Biosciences) and eluted with 1% trifluoroacetic acid prior to HPLC analysis. To follow the time course of LCI unfolding, native LCI was dissolved in the same buffer containing 6, 7, or 8 m GdnHCl. At given incubation times, aliquots were removed, quenched with an equal volume of 4% trifluoroacetic acid, and analyzed by HPLC. Heat denaturation was performed by submitting aliquots of the sample in Tris-HCl buffer (0.1 m, pH 8.4) containing 0.25 mm 2-mercaptoethanol to increasing temperatures of 45, 55, and 65 °C for 1 h and finally quenching the sample with equal volumes of 4% trifluoroacetic acid. The fluorescence spectra of LCI were measured with a 650–40 spectrofluorometer (PerkinElmer) scanning from 300 to 450 nm with excitation at 280 nm. LCI was dissolved in Tris-HCl buffer (0.1m, pH 8.4) containing 0.25 mm 2-mercaptoethanol and selected concentrations of denaturants (urea and GdnHCl). Denaturation of LCI was performed at 23 °C for 20 h. The final concentration of LCI was 2 μm. Fluorescence intensities of blank samples containing equivalent concentrations of the denaturant were also analyzed and subtracted from readings of the LCI samples. Isolated fractions of scrambled LCI (20 μg) were treated with 2 μg of thermolysin (Sigma, P-1512) in 30 μl of N-ethylmorpholine/acetate buffer (50 mm, pH 6.4). Digestions were carried out for 16 h at 37 °C for species X-LCI-g and at 50 °C for X-LCI-a. The reaction products were then fractionated by HPLC and analyzed by amino acid sequencing and mass spectrometry to identify the disulfide-containing peptides. The amino acid sequence of disulfide-containing peptides was analyzed by automated Edman degradation using a PerkinElmer Procise sequencer (model 494) equipped with an on-line phenylthiohydantoin-derivate analyzer. The molecular mass of disulfide-containing peptides was determined by MALDI-TOF mass spectrometry (Bruker Biflex TOF spectrometer equipped with a nitrogen laser with an emission wavelength of 337 nm). Denaturation of native LCI is defined by the conversion of the native structure to scrambled isomers. The denaturation curve was therefore generated by plotting the percentage of native LCI converted to scrambled isomers under increasing concentrations of a selected denaturant. In contrast, unfolding describes the state of the denatured LCI and is structurally defined by the composition of scrambled isomers. The LCI unfolding curves are determined by the relative concentrations of different scrambled isomers that exist along the unfolding pathway under increasing concentrations of a selected denaturant. Calculation of the yield of scrambled isomers was based on peak area integration. The data shown have a S. D. of ±5%. Native LCI was allowed to denature and unfold to form scrambled isomers in the presence of a thiol initiator and increasing concentrations of urea, GdnHCl, and GdnSCN. The HPLC profiles of the different states of unfolding are displayed in Fig. 1. Though it is not possible to determine how many scrambled isomers of LCI of the 104 possible ones populate the unfolded state, 9 of them, which amount to 90% of total denatured LCI, can be distinguished. They appear marked alphabetically in Fig. 1(a–i). The denaturation curves, calculated from the percentage of LCI converted to scrambled isomers, are shown in Fig. 2. Unfolding of LCI cooperatively occurs at 2–3 m GdnSCN with a midpoint at 2.4 m and at 3–4 m GdnHCl with a midpoint at 3.6 m. Based on the concentration that is required to achieve the same extent of denaturation, GdnSCN is about 1.5-fold more potent than GdnHCl. In contrast, urea is unable to denature the native LCI, because even at high concentration (8 m) the scrambled species are undetectable. Although urea is normally expected to be a weaker denaturant than GdnHCl (11Pace C.N. Methods Enzymol. 1986; 131: 266-280Crossref PubMed Scopus (2424) Google Scholar), LCI represents an extreme case in resistance to urea denaturation because even PCI, one of the more stable of the disulfide-containing proteins studied so far against urea denaturation, is about 50% denatured in 8 m urea (9Chang J.-Y., Li, L. Canals F. Avilés F.X. J. Biol. Chem. 2000; 275: 14205-14211Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).Figure 2LCI denaturation curves. The denatured fraction is expressed as the percentage of LCI that is converted to scrambled isomers. The denaturants are GdnSCN (▴), GdnHCl (♦), and urea (●). Denaturation was carried out at 20 °C for 20 h in Tris-HCl buffer (0.1 m, pH 8.4) containing 2-mercaptoethanol (0.25 mm) and the indicated concentrations of denaturant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Given the previous results obtained with the urea treatment and to ensure that no scrambled species hypothetically generated could have remained undetected, the behavior of the molecule upon urea and GdnHCl treatment was followed by fluorescence measurements. LCI contains two tryptophan residues at sequence positions 42 and 50. LCI exhibits increased fluorescence intensities when treated with increasing urea concentrations, albeit without any concomitant redshift. In contrast, treatment with 8 m GdnHCl generates a spectrum with decreased intensity and a clear redshift from 354 to 360 nm (Fig. 3). Since a displacement of the maximum of fluorescence, rather than a change in intensity, indicates denaturation, this observation supports the previously drawn conclusion that urea is unable to denature LCI. LCI was treated with increasing concentrations of methanol, allowing the reaction to reach equilibrium in 48 h. No denaturation at all was observed even at 65% methanol (data not shown). The same result was found when applying a standard heat denaturation procedure. Even at 65 °C with an incubation time of 1 h, no conversion of native LCI into denatured species was observed, supporting a previous observation of resonances related to the presence of three-dimensional structure by NMR at high temperatures (1Reverter D. Vendrell J. Canals Q. Horstmann J. Avilés F.X. Fritz H. Sommerhoff C.P. J. Biol. Chem. 1998; 273: 32927-32933Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The pathway of LCI unfolding can be represented by plotting the relative concentrations of the scrambled isoforms of denatured LCI along the process. The data corresponding to the evolution of the nine scrambled LCI isomers in the presence of increasing concentrations of GdnHCl and GdnSCN are shown in Fig. 4. Because of the almost complete inability of urea to unfold LCI, the unfolding curves are not shown for this condition. The relative concentrations of X-LCI-a and X-LCI-b correlate with the strength of the denaturing conditions in both cases. This phenomenon is more easily observed in GdnSCN curves where an increase in the relative concentration of X-LCI-a from 10 to 45% is observed within the interval of 2–6 m GdnSCN, while an increase from 15 to 37% is observed within the interval of 3–6m GdnHCl. To a lower extent X-LCI-b shows the same phenomenon. X-LCI-f, X-LCI-g, and X-LCI-h show an inverse correlation between their relative abundances and the denaturing conditions. X-LCI-g shows the biggest differences (25–26% in a range of 1–6m GdnSCN). Among these three species, X-LCI-g is the most highly populated scrambled at low concentrations of GdnHCl (16%) and GdnSCN (25%). X-LCI-g is followed by X-LCI-f and X-LCI-h (both 14% in GdnSCN and 12 and 8% in GdnHCl, respectively). According to these results, it is possible to separate the scrambled isomers of LCI into two groups, which suffer a different kind of evolution along the unfolding pathway. Two species, X-LCI-a and X-LCI-g, which represent each population and show the higher relative concentration at extremes of denaturant concentration, were selected to further study their disulfide pairing and be structurally characterized. Along the unfolding pathway of LCI, 9 major fractions can be identified as scrambled isomers, distinguishable from a population of 104 possible isoforms (Fig. 1). They were separated by reverse-phase HPLC with a linear acetonitrile gradient. Two fractions, X-LCI-a and X-LCI-g, selected according to the aforementioned criteria, were further isolated and analyzed to determine their disulfide structures. After digestion with thermolysin, peptides were isolated by HPLC and characterized by Edman sequencing and mass spectrometry to identify the disulfide pairing. Table I summarizes the sequence and mass spectrometry analyses of the peptides isolated after digestion of the two scrambled isomers.Table IStructures of disulfide-containing peptides derived from thermolysin digestion of scrambled LCIPeptideSequenceSequence positionCys–CysMr found (exp.)a-4LCYQPDQVC10–18Cys11–Cys181066.22 (1067.22)a-9GECNPHPTAPWCREGAVEWVP32–52Cys34 –Cys432333.59 (2333.09)TGQCRTTCIPYV55–66Cys58–Cys621339.56 (1339.61)ECNPHPTAPWC33–43Cys34–Cys431252.40 (1252.16)a-10AAPLPSEGECNPHPTAPWCREG25–46Cys34 –Cys432317.55 (2316.51)a-12DESFLCYQPDQVCCFICRGA6–25Cys11–Cys182293.61 (2294.56)Cys19–Cys22a-13DESFLCYQPDQVCCFICRGAAPLPSEGEC6–57Cys11 –Cys185740.39 (5739.97)NPHPTAPWCREGAVEWVPYSTGQCys19 –Cys22Cys34 –Cys43g-5CFI19–21Cys19–Cys341866.06 (1865.34)APLPSEGECNPHPTA26–40g-8CYQ11–13Cys11–Cys621328.58 (1328.72)RTTCIPYVE59–66WCREGAVEWVPYSTGQCRTT42–61Cys43–Cys582329.60 (2329.99)GAAPLPSEGECNP24–36Cys341241.34 (1241.27)g-9PDQVCCFICRGAAPLPSEGECN14–35Cys18 –Cys222309.65 (2309.94)Cys19 –Cys34g-11PWCREGAVEWVPYSTGQCRTT41–61Cys43 –Cys582426.72 (2426.39)Peptides a-n and g-n correspond, respectively, to thermolysin digestion products of X-LCI-a and X-LCI-g. The complete sequences of each peptide are shown, and the residues analyzed by Edman degradation are underlined. Sequence numbering refers to the recombinant LCI that contains a construction-added Gly as the N-terminal residue. Open table in a new tab Peptides a-n and g-n correspond, respectively, to thermolysin digestion products of X-LCI-a and X-LCI-g. The complete sequences of each peptide are shown, and the residues analyzed by Edman degradation are underlined. Sequence numbering refers to the recombinant LCI that contains a construction-added Gly as the N-terminal residue. X-LCI-a was clearly identified as the beads-form isomer (contains Cys11–Cys18, Cys19–Cys22, Cys34–Cys43, and Cys58–Cys62 bonds) (Fig. 5) from the unambiguous results summarized in Table I. The structure of X-LCI-a was deduced from the identification of three major thermolytic peptides, a-4, a-10, and a-9, that showed an unique possible Cys pairing (Table I) consistent with sequencing and mass spectrometry. X-LCI-g adopts Cys11–Cys62, Cys18–Cys22, Cys19–Cys34, and Cys43–Cys58 pairing (Fig. 5). From the unambiguous analysis of peptide g-11, and according to its determined Mr (2426.39), Cys43–Cys58 was the first disulfide pair assigned. The analysis of g-5 showed two separated sequences bound by a unique disulfide pair, Cys19–Cys34. Both disulfide pairs helped to further define the pairing of peptides g-8 and g-9, whereas only one disulfide pair could fit out of a mixture of possibilities. Given that GdnHCl concentration was found to be proportional to the amount of denatured PCI in terms of accumulated scrambled species, the kinetics of unfolding of the native LCI was studied at different denaturant strengths (Fig. 6). The unfolding of LCI is complete in 120 min at 8 and 7 m GdnHCl, whereas it takes 300 min to complete in 6 m GdnHCl. A significant difference can thus be achieved by slightly modifying the concentration of certain denaturant. The rate constant of unfolding at 8 and 7m GdnHCl (3.3 × 10−3 min−1) is about 2.5-fold greater than that observed at 6 m GdnHCl (8.3 × 10−3 min−1). The denaturation curves obtained by representing the fraction of native LCI converted into the scrambled species under increasing concentrations of denaturants (chaotropic agents, organic solvents, and temperature) are indicative of the conformational stability of LCI. GdnHCl and GdnSCN denature LCI quantitatively in a cooperative manner at concentrations between 2–3 and 3–4 m, respectively, in accordance with the observations made in the case of cardiotoxin III (CTX-III) (12Chang J.-Y. Kumar T.K.S. Yu C. Biochemistry. 1998; 37: 6745-6751Crossref PubMed Scopus (19) Google Scholar), an all β-sheet protein. These values are higher than those obtained for PCI, another disulfide-containing carboxypeptidase inhibitor (9Chang J.-Y., Li, L. Canals F. Avilés F.X. J. Biol. Chem. 2000; 275: 14205-14211Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) that is almost devoid of any regular secondary structure, (13Rees D.C. Lipscomb W.N. J. Mol. Biol. 1982; 160: 475-498Crossref PubMed Scopus (247) Google Scholar, 14Clore G.M. Groneborn A.M. Nilges M. Ryan C.A. Biochemistry. 1987; 26: 8012-8023Crossref PubMed Scopus (264) Google Scholar). The higher concentration of denaturants required for LCI could reflect the contribution of secondary structure to the stability of the LCI fold. The higher stability of LCI compared with PCI is also shown by the fact that urea is unable to denature LCI, whereas PCI reaches 50% denaturation at 8 m urea (9Chang J.-Y., Li, L. Canals F. Avilés F.X. J. Biol. Chem. 2000; 275: 14205-14211Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). At this point it should be mentioned that the increase in fluorescence intensity observed in urea-treated LCI (Fig. 3) is not indicative of denaturation, because no significant red shift is observed between 0 and 8 m. However, a red shift (from 354 to 360 nm) is clearly observed in the 8m GdnHCl-denatured LCI, which additionally shows a decreased fluorescence intensity. Thus, the increase in fluorescence intensity in urea-treated samples may be indicative of a different hydrophobic environment of tryptophan residues upon urea treatment generated by the dynamic relocation of secondary structure elements. PCI, although less stable than LCI, is still a very stable fold with its globular structure stabilized by a compact hydrophobic core and by three disulfide bridges forming a T-knot. Both carboxypeptidase inhibitors are designed to resist extreme external conditions and still be functional. The comparison between the stabilities of other disulfide-containing proteins studied by this method is shown in Table II and is expressed as the concentration of denaturant required to achieve 50% of denaturation of the protein. The following two crucial aspects of these data obtained with seven different disulfide-containing proteins need to be mentioned. 1) The extent of denaturation achieved by each fold is dependent upon the type of denaturant. GdnHCl is generally more potent than urea, albeit the relative potencies vary from protein to protein. The two extreme examples correspond to LCI (in which urea is unable to quantitatively unfold LCI) and TAP, where urea is more potent than GdnHCl. The clear difference of potency between urea and GdnHCl has been extensively reported and can be attributed to the differential mode of action between these denaturants, although the detailed mechanisms are not yet fully understood (15Tanford C. Adv. Protein Chem. 1968; 23: 121-282Crossref PubMed Scopus (2437) Google Scholar, 11Pace C.N. Methods Enzymol. 1986; 131: 266-280Crossref PubMed Scopus (2424) Google Scholar, 16Pace C.N. Laurents D.V. Thomson J.A. Biochemistry. 1990; 29: 2564-2572Crossref PubMed Scopus (351) Google Scholar, 17Liepinsh E. Otting G. J. Am. Chem. Soc. 1994; 116: 9670-9674Crossref Scopus (107) Google Scholar). It is known that both denaturants disrupt noncovalent interactions that stabilize the native fold (18Dill K.A. Shortle D. Annu. Rev. Biochem. 1991; 60: 795-825Crossref PubMed Scopus (911) Google Scholar), mainly hydrophobic interactions, by causing water to become a better solvent for nonpolar amino acids. In addition to this common property, GdnHCl is a salt and also suppresses electrostatic interactions. The suppression of electrostatic interactions is fundamental to achieve unfolding of an all β-sheet protein, as is the case for LCI. 2) LCI is globally the more stable protein studied by this method as yet. In addition to the inability of urea to denature it, no denaturation was observed either with 65% methanol or by heat treatment (incubation at 65 °C for 1 h). The high stability of LCI arises from an optimal combination of disulfide-bonding, a compact hydrophobic core, and the high percentage of regular secondary structure. This high conformational stability of LCI in extreme conditions would greatly facilitate its application to human therapy as a profibrinolytic agent.Table IIConcentration of different denaturants required to achieve 50% of denaturation of several disulfide-containing ProteinsProteinGdnSCNGdnHClUreamLCI2.43.6No denaturationPCI0.71.45>8RNase A0.752.255.75TAP1.04.24.0IGF-11.53.25.5Hirudin25>8CTX-IIINot determined2.33.4These data were determined by the method of disulfide scrambling between the native and scrambled isomers. The data has a S.D. ± 5%. The references are described in the text. Open table in a new tab These data were determined by the method of disulfide scrambling between the native and scrambled isomers. The data has a S.D. ± 5%. The references are described in the text. The denatured LCI was found to be composed of at least 9 scrambled isomers out of the possible 104 (Fig. 1). The unfolding pathway of LCI was determined by the evolution of the scrambled isomers generated under increasing concentrations of GdnHCl and GdnSCN (Fig. 4). Although the absolute amounts of each scrambled in the unfolding pathway is characteristic of the denaturant assayed, the composition and evolution of the different scrambled species is comparable. This indicates that these scrambled species correspond to the main transient intermediates in the unfolding pathway of LCI. Since for an unfolding reaction the strong denaturation condition precludes the accumulation of any intermediate (19Matouschek A. Serrano L. Fersht A.R. J. Mol. Biol. 1992; 224: 819-835Crossref PubMed Scopus (202) Google Scholar), the characterization of unfolding intermediates is only possible in disulfide-containing proteins. There are two populations of scrambled species that undergo variations along the unfolding pathway. One accumulates under strong denaturing conditions. This was the case for X-LCI-a and to a lesser extent for X-LCI-b. The other population shows an inverse correlation between their relative abundances and the denaturing conditions. This is the case for X-LCI-f, X-LCI-h, and X-LCI-g, the latter being the most populated scrambled at low concentrations of denaturant. Following previous results obtained with PCI (9Chang J.-Y., Li, L. Canals F. Avilés F.X. J. Biol. Chem. 2000; 275: 14205-14211Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), TAP (8Chang J.-Y. J. Biol. Chem. 1999; 274: 123-128Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), and insulin-growth factor (IGF-1) (10Chang J.-Y. Marki W. Lai P.H. Protein Sci. 1999; 8: 1463-1468Crossref PubMed Scopus (18) Google Scholar), an open or relaxed structure is expected to be found for the first population (including X-LCI-a and X-LCI-b) that could explain the prevalence of both forms in high concentration of denaturants. In contrast, the population represented by X-LCI-g should have another kind of non-native structure that turns unstable at high denaturant strength. These non-native structures are likely to be more compact than the unfolded state. The assignment of the disulfide structure of the most abundant scrambled species within each population has confirmed these hypotheses (Fig. 5). The identification of X-LCI-a as the beads-form isomer was already expected from similar results found with other proteins, such as PCI (9Chang J.-Y., Li, L. Canals F. Avilés F.X. J. Biol. Chem. 2000; 275: 14205-14211Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), TAP (8Chang J.-Y. J. Biol. Chem. 1999; 274: 123-128Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), and IGF-1 (10Chang J.-Y. Marki W. Lai P.H. Protein Sci. 1999; 8: 1463-1468Crossref PubMed Scopus (18) Google Scholar), which also show a predominance of the bead-form isomer under strong denaturing conditions. This can be due to a common pathway of unfolding where the polypetide chain undergoes a progressive expansion and relaxation toward the shape of linear structure. In contrast, X-LCI-g is an unstable intermediate with a quite compact non-native structure, since the N- and C- terminal ends of the molecule have already been brought together, as suggested by the pairing of disulfide bonds. The rate constant for unfolding of LCI at 7 and 8 m GdnHCl was found to be very similar, whereas it is about 2.5-fold lower at 6 m. Thus, a significant difference can be achieved by slightly modifying the concentration of certain denaturant. On the other hand, the variation of the rate constant of unfolding upon increasing concentrations of denaturants is not linear. This is in contrast not only with the behavior of proteins lacking disulfide-bonding (19Matouschek A. Serrano L. Fersht A.R. J. Mol. Biol. 1992; 224: 819-835Crossref PubMed Scopus (202) Google Scholar) but also with the unfolding kinetics of CTX-III (12Chang J.-Y. Kumar T.K.S. Yu C. Biochemistry. 1998; 37: 6745-6751Crossref PubMed Scopus (19) Google Scholar) in which there is a linear decrease of the rate constant for unfolding under denaturation by the same denaturant. The abrupt change in the rate constant for unfolding in LCI between 6 and 7 m GdnHCl could be an indication of the cooperative nature of the process and points out the relevance of hydrogen-bonding (regular secondary structure) in maintaining the native structure of LCI. On the other hand, the kinetics of LCI unfolding is about 15–25-fold slower than that of CTX-III (12Chang J.-Y. Kumar T.K.S. Yu C. Biochemistry. 1998; 37: 6745-6751Crossref PubMed Scopus (19) Google Scholar). This could be one of the explanations for the high stability of LCI, although a refolding kinetics analysis is needed to confirm this hypothesis. The results presented in this study confirm that LCI fulfills the basic requirements of high stability and slow kinetics of unfolding that are expected for a protein molecule with potential applications in human therapy." @default.
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• W1984406971 title "The Unfolding Pathway of Leech Carboxypeptidase Inhibitor" @default.
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