Báo cáo khoa học: Oxidative folding of conotoxins sharing an identical disulfide bridging framework ppt

Oxidative folding of conotoxins sharing an identical disulfide bridging framework Erika Fuller 1 , Brad R. Green 1 , Phil Catlin 1 , Olga Buczek 2 , Jacob S. Nielsen 1 , Baldomero M. Olivera 2 and Grzegorz Bulaj 1,2 1 Cognetix Inc, Salt Lake City, UT, USA 2 Department of Biology, University of Utah, Salt Lake City, UT, USA Conotoxins form a diverse group of disulfide-rich peptide neurotoxins, comprising an estimated 50 000– 100 000 unique sequences that are produced by Conus snails (Fig. 1A) (reviewed most recently in [1]). In con- trast to this vast diversity of conotoxin primary amino acid sequences, these peptides can be classified into only a handful of structural classes. These classes (also called families) are distinguished based on disulfide frameworks, determined by the number and pattern of cysteine residues and the native-like pairing of disulfide bridges that stabilize the biologically active conforma- tion. Figure 1B shows examples of conotoxins with Keywords conotoxin; oxidative folding; disulfide bonds Correspondence G. Bulaj, Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA Fax: +1 801 585 5010 Tel: +1 801 581 8370 E-mail: bulaj@biology.utah.edu (Received 6 December 2004, revised 2 February 2005, accepted 8 February 2005) doi:10.1111/j.1742-4658.2005.04602.x Conotoxins are short, disulfide-rich peptide neurotoxins produced in the venom of predatory marine cone snails. It is generally accepted that an estimated 100 000 unique conotoxins fall into only a handful of structural groups, based on their disulfide bridging frameworks. This unique mole- cular diversity poses a protein folding problem of relationships between hypervariability of amino acid sequences and mechanism(s) of oxidative folding. In this study, we present a comparative analysis of the folding properties of four conotoxins sharing an identical pattern of cysteine resi- dues forming three disulfide bridges, but otherwise differing significantly in their primary amino acid sequence. Oxidative folding properties of M-superfamily conotoxins GIIIA, PIIIA, SmIIIA and RIIIK varied with respect to kinetics and thermodynamics. Based on rates for establishing the steady-state distribution of the folding species, two distinct folding mecha- nisms could be distinguished: first, rapid-collapse folding characterized by very fast, but low-yield accumulation of the correctly folded form; and sec- ond, slow-rearrangement folding resulting in higher accumulation of the properly folded form via the reshuffling of disulfide bonds within folding intermediates. Effects of changing the folding conditions indicated that the rapid-collapse and the slow-rearrangement mechanisms were mainly deter- mined by either repulsive electrostatic or productive noncovalent inter- actions, respectively. The differences in folding kinetics for these two mechanisms were minimized in the presence of protein disulfide isomerase. Taken together, folding properties of conotoxins from the M-superfamily presented in this work and from the O-superfamily published previously suggest that conotoxin sequence diversity is also reflected in their folding properties, and that sequence information rather than a cysteine pattern determines the in vitro folding mechanisms of conotoxins. Abbreviations ACN, acetonitrile; BPTI, bovine pancreatic trypsin inhibitor; Fmoc, N-(9-fluorenyl)methoxycarbonyl; GIIIA, l-conotoxin GIIIA; GSH, reduced glutathione; GSSG, oxidized glutathione; H ⁄ D, hydrogen ⁄ deuterium; MTBE, methyl-tert butyl ether; PIIIA, l-conotoxin PIIIA; PDI, protein disulfide isomerase; RIIIK, jM-conotoxin RIIIK; SmIIIA, l-conotoxin SmIIIA; TFA, trifluoroacetic acid. FEBS Journal 272 (2005) 1727–1738 ª 2005 FEBS 1727 relatively little amino acid sequence homology (with the exception of cysteine residues) that share the same disulfide bridging frameworks. Molecular diversity of conotoxins presents a unique protein folding paradox. If there is such hypervaria- bility of primary amino acid sequences within each disulfide scaffold, what structural features determine the formation of the native disulfide bridges that sta- bilize the properly folded conformation? Is the distri- bution of cysteine residues sufficient to determine formation and rearrangement of disulfide bonds lead- ing to the correctly folded species? The conotoxin folding problem was previously illustrated using cono- toxins belonging to the O-superfamily (Fig. 1 in [2]). Several folding studies on conotoxins within the same family include x-conotoxins [3], a-conotoxins [4,5], d-conotoxins [6] and spasmodic peptides [7]. The main finding from these studies is that changes in the primary sequence significantly influence folding yields. For example, the work of Price-Carter et al. [3] showed that even relatively small changes in amino acid sequence, such as x-MVIIA and x-MVIIC, could lead to a threefold decrease in folding yields. However, none of the studies mentioned above inves- tigated in detail how folding kinetics and thermo- dynamics can vary among naturally occurring conotoxins that share an identical disulfide bridging framework. Conotoxins from the M-superfamily contain six cys- teine residues forming a characteristic three-disulfide bridging pattern (Fig. 1B) [8–12]. Additional post- translational modifications include 4-hydroxyproline and pyroglutamate. As shown in Table 1, these cono- toxins are characterized by a relatively large number of positively charged residues, previously shown to be important for biological activity [13–15]. To date, five Fig. 1. Molecular diversity of conotoxins. (A) Morphological diversity of cone snails from which the four conotoxins studied were isolated. From the top left, clockwise: Conus geographus, C. purpurascens, C. radiatus, C. stercusmuscarum (bar represents 10 mm). (B) Diversity of conotoxins and the ‘conotoxin folding puzzle’. Shown are several conotoxins sharing an identical disulfide bridging pattern but differing in their primary amino acid sequence. Hundreds of conotoxins differing in their primary amino acid sequences fall into each disulfide framework [49–52]. To emphasize distribution of some structural features, selected residues were color-coded as follows: positively charged residues, red; prolines or 4-hydroxyprolines (O), blue. Z, pyroglutamate; #, C-terminal amidation. Table 1. M-superfamily conotoxins isolated from fish-hunting cone snails and their molecular targets. #, C-terminal amidation; Z and O, pyroglutamate and 4-hydroxyproline, respectively. Name Conus species Sequence Net charge Target ion channel Ref GIIIA geographus RDCCTOOKKCKDRQCKOQRCCA# +6 Skeletal muscle Na channel [18,19] GIIIB geographus RDCCTOORKCKDRRCKOMKCCA# +7 Skeletal muscle Na channel [18] GIIIC geographus RDCCTOOKKCKDRRCKOLRCCA# +7 Skeletal muscle Na channel [18] PIIIA purpurascens ZRLCCGFOKSCRSRQCKOHRCC# +6 Neuronal ⁄ skeletal Na channel [20] SmIIIA stercusmuscarum ZRCCNGRRGCSSRWCRDHSRCC# +5 TTX-resistant Na channel [48] RIIIK radiatus LOSCCSLNLRLCOVOACKRNOCCT# +4 KV1.2 and Shaker K channel [16,17] Oxidative folding of conotoxins E. Fuller et al. 1728 FEBS Journal 272 (2005) 1727–1738 ª 2005 FEBS l-conotoxins, targeting sodium channels, and one jM-conotoxin, targeting potassium channels, have been characterized in detail [1,16,17]. l-Conotoxins GIIIA, GIIIB and GIIIC share 86% identity between their primary amino acid sequences [18]. However, there is very li ttle se quence similarity between l-conotoxin GIIIA (GIIIA), l-conotoxin PIIIA (PIIIA), l-conotoxin SmIIIA (SmIIIA) and jM-conotoxin RIIIK (RIIIK) when noncysteine residues are compared. Despite sequence diversity among these conotoxins, their three- dimensional model structures show overall structural similarities with some local conformational differences, in particular in the N-terminal portion of the mole- cules [8–12]. To compare folding kinetics and thermodynamics of conotoxins containing identical cysteine patterns, but otherwise differing in their amino acid sequence, we investigated the oxidative folding of GIIIA, PIIIA, SmIIIA and RIIIK. Distribution of folding species, folding kinetics and thermodynamics differed significantly among the four peptides. Two distinct folding mechanisms could be distinguished, namely rapid-collapse and slow-rearrangement folding. Our results suggest that conotoxin sequence diversity is also reflected in in vitro folding properties, and that cysteine patterns do not determine folding mecha- nisms of conotoxins. Results Oxidative folding of GIIIA, PIIIA, SmIIIA and RIIIK To examine how sequence hypervariability may affect in vitro folding properties of conotoxins, we selected four peptides from the M-superfamily, GIIIA, PIIIA, SmIIIA and RIIIK, which share an identical disulfide bridging framework, but differ significantly in their primary amino acid structure (Table 1 and Fig. 1B). In addition to sharing the M-superfamily bridging pat- tern (Cys I-Cys IV, Cys II-Cys V and Cys III-Cys VI), these peptides contain a C-terminal amidation, a com- mon post-translational modification among conotox- ins. PIIIA and SmIIIA also contain a pyroglutamate at their N-terminus. Several structural features can be distinguished among all four peptides: the lengths of individual loops (the number of amino acid residues between individual cysteines); distribution of charged residues; and the presence of prolines ⁄ hydroxyprolines. Among M-conotoxins, a relatively high number of positively charged residues results in a very basic character of the peptides with the net charge ranging from +5 for SmIIIA to +6 for GIIIA and PIIIA, and even +7 for GIIIB and GIIIC (Table 1). The peptides were synthesized on solid support using the standard Fmoc chemistry. The cysteine thiols were protected with trityl groups, which were removed simul- taneously with the cleavage of the peptide from the resin. The cleaved peptides were purified on preparative reversed-phase HPLC and dried by lyophilization. For folding experiments, the dry reduced peptides were resuspended in 0.01% (v ⁄ v) trifluoroacetic acid (TFA) and added to the folding mixtures containing 0.1 m Tris ⁄ HCl buffer pH 7.5, 0.1 mm EDTA and a mixture of 1 mm reduced glutathione (GSH) and 1 mm oxidized glutathione (GSSG). To quench the folding reaction, aliquots were withdrawn and transferred to tubes con- taining formic acid. The quenched folding mixtures were separated by analytical C 18 HPLC. For each peptide, the identity of the correctly folded species was deter- mined by comparing HPLC retention times of the folding products with the reference synthetic peptides prepared as described previously [11,16,19,20]. Representative HPLC separations of the folding mixtures quenched after 30 s, 10 min and 60 min for GIIIA, PIIIA, SmIIIA and RIIIK are shown in Fig. 2. Comparing the distribution of the folding products at the early and later folding stages indicated that the correctly folded RIIIK and GIIIA formed significantly slower, relative to a rapid accumulation of the folded PIIIA and SmIIIA. For RIIIK and GIIIA, the prop- erly folded species were almost undetectable after 30 s, but their final folding yields exceeded 60% and 30%, respectively. The RIIIK folding was very cooperative, with relatively low-level accumulation of the transient folding intermediates. GIIIA folding was characterized by a transient accumulation of a number of folding products. For both PIIIA and SmIIIA, the overall dis- tribution of the folding species did not differ signifi- cantly between 30 s and 60 min. In contrast to RIIIK and GIIIA, the correctly folded forms of PIIIA or SmIIIA were found to accumulate early during fold- ing. However, a large number of additional folding species were detectable for these two peptides, even at the later folding stages. To determine whether the accumulated products represented fully oxidized spe- cies, HPLC peaks from the 60 min folding samples were collected, dried and analyzed by MALDI-TOF. The determined molecular masses of the peaks corres- ponding to the folded forms agreed with the calculated masses: RIIIK [MH + ]exp ¼ 2649.0, [MH + ]calc ¼ 2648.7; GIIIA [MH + ]exp ¼ 2608.7, [MH + ]calc ¼ 2608.5; PIIIA [MH + ]exp ¼ 2604.3, [MH + ]calc ¼ 2604.2; SmIIIA [MH + ]exp ¼ 2605.0, [MH + ]calc ¼ 2605.5. For each conotoxin, a majority of the other accumulated folding species exhibited molecular mas- ses similar to the fully oxidized species within one mass E. Fuller et al. Oxidative folding of conotoxins FEBS Journal 272 (2005) 1727–1738 ª 2005 FEBS 1729 unit, suggesting that they represented misfolded prod- ucts, rather than folding intermediates or mixed disul- fide forms. For each conotoxin, the time course for the appear- ance of the correctly folded species was analyzed and fit to the pseudo first-order kinetics (Fig. 3). Extending folding time beyond two hours for all four conotoxins did not result in changes in distribution and accumula- tion of the folding products. The apparent rate con- stants obtained from the kinetic fits were as follows: 0.05 min )1 for RIIIK, 0.04 min )1 for GIIIA, 3.3 min )1 for PIIIA and 3.8 min )1 for SmIIIA. In contrast to differences in the rates for forming the correctly folded species, the time-course of the disappearance of the reduced form was comparable for all four peptides. This is illustrated in Fig. 3 (insets), where the apparent rates for the disappearance of the reduced form were obtained from the fit: 2.5 min )1 for RIIIK, 3.3 min )1 for GIIIA, 4.7 min )1 for PIIIA and 6.6 min )1 for SmIIIA. Based on these results, at least two types of folding mechanisms could be distinguished: (a) the slow-rearrangement folding, best represented by GIIIA and RIIIK, where formation of the correctly folded species was significantly slower than the formation of folding intermediates; and (b) rapid-collapse folding, as observed for SmIIIA and PIIIA, where the steady- state accumulation of the correctly folded form was rapidly established within the first few minutes of the folding reaction. The slow-rearrangement was charac- terized by relatively higher folding yields, as compared to the rapid-collapse folding. Folding determinants for GIIIA and SmIIIA To characterize factors that determine the two folding mechanisms, rapid-collapse (SmIIIA and PIIIA) and slow-rearrangement (RIIIK and GIIIA), we further explored the folding of the two conotoxins GIIIA and SmIIIA under several different conditions. The folding reactions were quenched after two (SmIIIA) or four (GIIIA) hours, and the steady-state accumulation of correctly folded GIIIA and SmIIIA was determined from HPLC analysis. The effects of changing redox conditions, pH, increasing denaturing conditions (4 m urea or 40 °C) or increasing ionic strength to 1 m NaCl were studied, as illustrated in Fig. 4. Neither more reducing or oxidizing conditions at either pH 7.5 or 8.7 resulted in significant improvement of the fold- ing yields for GIIIA or SmIIIA. At pH 7.5, changing the redox conditions from 1 mm GSSG ⁄ 1mm GSH to 1mm GSSG ⁄ 5mm GSH caused a twofold decrease in the accumulation of both peptides, suggesting a com- parable stability of the native disulfide bonds in SmIIIA and GIIIA. GIIIA folding was very sensitive to the increased temperature or denaturant. In con- trast, changing temperature or urea did not signifi- cantly affect the steady-state distribution of the SmIIIA folding species. As GIIIA and SmIIIA contain a relatively high number of positively charged residues, we examined the effects of high ionic strength on their folding prop- erties. The electrostatic effects were previously shown to play a role in glutathione-assisted oxidative folding Fig. 2. Oxidative folding of conotoxins GIIIA, PIIIA, SmIIIA and RIIIK. Folding reactions were carried out in 0.1 M Tris ⁄ HCl, pH 7.5 in the presence of 1 m M GSSG and 1 mM GSH at the ambient temperature (23–25 °C). Reactions were quenched by acidification, and the aliquots were subjected to HPLC analysis as described in the Experimental procedures section. Retention time of the correctly folded form was determined by HPLC analysis of the reference cono- toxins prepared as described previously [11,16,19,20]. Oxidative folding of conotoxins E. Fuller et al. 1730 FEBS Journal 272 (2005) 1727–1738 ª 2005 FEBS [21]. As illustrated in Fig. 4, the presence of 1 m NaCl strongly influenced the folding of SmIIIA, but had a somewhat smaller effect on GIIIA folding. Interest- ingly, the high ionic strength improved folding of PIIIA but not RIIIK (data not shown). High concen- trations of selected osmolytes (20% glycerol, 1 m sucrose, 200 mm betaine, or 500 mm sarcosine) did not affect accumulation of GIIIA, PIIIA or SmIIIA (data not shown). Protein disulfide isomerase (PDI) is a key enzyme catalyzing the rearrangement of protein disulfide bonds [22,23]. To determine how PDI could affect the folding of slowly-rearranging GIIIA as compared to that of the rapidly-accumulating SmIIIA, we employed folding conditions as shown previously for a-conotoxin GI [2]. The reactions were carried out with 0.1 mm GSSG, pH 7.5, where the oxidative folding is significantly slower compared to that at 1 mm GSSG ⁄ 1mm GSH. Figure 5 shows time-courses of the appearance of the correctly folded SmIIIA and GIIIA in the presence and absence of PDI. The addition of the enzyme accel- erated the accumulation of the correctly folded GIIIA, but did not affect SmIIIA folding. The apparent lag phase in appearance of the correctly folded GIIIA was consistent with the predominant rearrangement as a rate-limiting step in efficient folding of GIIIA. These results confirmed a key role of PDI in catalyzing the reshuffling steps, consistent with previous observations with a-conotoxin GI [2]. Probing conformational properties of GIIIA and SmIIIA To examine whether different folding mechanisms of SmIIIA and GIIIA could be accounted for by differ- ences in their conformational flexibility, we employed MALDI-TOF analysis of hydrogen ⁄ deuterium (H ⁄ D) exchange of the correctly folded and reduced forms of these peptides. H ⁄ D exchange monitored by mass spectrometry is a well-established technique to probe conformational properties of peptides and proteins [24,25]. The deuterated phosphate buffer, pH 7.0 (pH not corrected for the isotopic effect) was used to allow exchange of protons over a 24 h time-course at ambi- ent temperature (23–25 °C). The H ⁄ D exchange reac- tion was quenched by mixing aliquots with chilled matrix solution, pH 2.5, followed by immediately des- iccating the mixture and analyzing by MALDI-TOF. The H ⁄ D exchange for the reduced forms was carried out as a control experiment to verify D-mass for all exchangeable protons. Following the exchange reac- tion, aliquots were additionally analyzed on HPLC to Fig. 3. Folding kinetics of conotoxins GIIIA, PIIIA, SmIIIA and RIIIK at pH 7.5 in the presence of 1 m M GSSG and 1 mM GSH. Folding reactions were performed as shown in Fig. 2. HPLC peaks were integrated and used to plot a time-course of the appearance of the properly folded conotoxins (main plots) and a disappearance of the reduced forms (insets). The experimental points were averaged from three independent experi- ments and fit to the first-order equation; values from these fits are given in the Results section. Note that although correctly folded RIIIK was a predominant product, a large number of folding species that accumu- lated at a very low level accounted for 40% of the final folding products. E. Fuller et al. Oxidative folding of conotoxins FEBS Journal 272 (2005) 1727–1738 ª 2005 FEBS 1731 verify that no significant oxidation of the Cys residues occurred during the time-course of the exchange reac- tion. Figure 6 shows the results of the H ⁄ D exchange experiments. The D-mass for the reduced forms of GIIIA and SmIIIA was 40 and 45, respectively, and did not significantly change between 1 and 5 min. The correctly folded GIIIA and SmIIIA exchanged 39 and 40 labile protons, respectively. The D-mass did not sig- nificantly vary between 1 min and 24 h of reaction time, suggesting a very rapid exchange, similar to that observed for the reduced form. The quantitative exchange of labile protons observed in the reduced and correctly folded forms of GIIIA suggested a lack of slow-exchanging core and ⁄ or rap- idly exchanging conformations. The difference of five protons that did not exchange in the correctly folded SmIIIA, as compared to the reduced form, could be accounted for by a more stable backbone conforma- tion relative to GIIIA. It is conceivable that differences in the H ⁄ D exchange kinetics between GIIIA and SmIIIA could occur within the first minute of the reac- tion, but technical limitations of MALDI-TOF-monit- ored H ⁄ D exchange precluded exploration of such early time points. Discussion Due to their unprecedented molecular diversity, cono- toxins appear to be very useful tools in studying the protein folding problem; more specifically, in addres- sing the question of what structural factors determine folding mechanisms of polypeptides sharing little sequence homology. In this study, we examined the Fig. 4. Effects of folding environment on the accumulation of the cor- rectly folded species in GIIIA and SmIIIA. Folding reactions were per- formed under identical conditions as described in Fig. 2 (control). Ratios represent concentrations of oxidized and reduced glutathione, respectively. Relative to the folding with 1 m M GSSG ⁄ 1mM GSH, 25 °C, pH 7.5 (‘control’), the following folding conditions were chan- ged: temperature 40 °Cor4 M urea or 1 M NaCl. The steady-state accumulation of the correctly folded species was determined after two or four hours of the folding reaction for SmIIIA and GIIIA, respectively. Bar graphs represent folding yields, relative to the accu- mulation of the other folding species. Error bars represent standard error calculated from three independent experiments. Fig. 5. Effect of protein disulfide isomerase on the oxidative folding of SmIIIA and GIIIA. Folding reactions were carried out in 0.1 M Tris ⁄ HCl, pH 7.5 in the presence of 0.1 mM GSSG at the ambient temperature (23–25 °C). Bovine protein disulfide isomerase (2 l M) was added prior to addition of the reduced forms of conotoxins. The appearance of the correctly folded species was determined by HPLC as shown in Fig. 3. U nfilled circles denote absence of PDI; filled circles denote the reaction with the enzyme. Oxidative folding of conotoxins E. Fuller et al. 1732 FEBS Journal 272 (2005) 1727–1738 ª 2005 FEBS oxidative folding of four conotoxins, GIIIA, PIIIA, SmIIIA and RIIIK, sharing identical disulfide bridging framework, but differing in their amino acid sequence. Folding of SmIIIA and PIIIA occurred in the rapid- collapse fashion, resulting in relatively lower yields, as compared to GIIIA and RIIIK, which folded through the slow-rearrangement mechanism. Optimization of folding yields for GIIIA and SmIIIA by changing redox conditions (GSSG ⁄ GSH) confirmed their margi- nal stability under in vitro conditions. Our results sug- gest that the pattern of cysteine residues forming the disulfide framework does not provide sufficient infor- mation in determining the folding mechanism or thermodynamic stability. This work is the second com- prehensive study of the structure–folding relationships in conotoxins. In the original study by Price- Carter et al. [3,26], the authors analyzed oxidative folding of five x-conotoxins (O-superfamily), as shown in Table 2. The steady-state accumulation of the cor- rectly folded species varied among these conotoxins, ranging from 16% for MVIIA to 50% for three cono- toxins MVIIA, GVIA and SVIA. The authors conclu- ded that despite limited sequence conservation, at least some conotoxins can be effectively folded, and that noncovalent interactions played an important role in the correct folding of these peptides. Limited results of structure–folding relationships were also presented for d-conotoxins [6]. Conotoxins from this family are char- acterized by poor folding yields resulting from their hydrophobic nature [6,27,28]. Our work is in accord with the previous observations that sequence diversity among conotoxins is also reflected in their in vitro folding properties. There is an intriguing analogy between sequence– folding relationships for conotoxins studied here and those characterized previously for disulfide-coupled fol- ding of bovine pancreatic trypsin inhibitor (BPTI). The folding mechanism of BPTI was extensively studied using mutational analysis [29–34]. The BPTI folding may represent an example of the slow-rearrangement Fig. 6. Kinetics of H ⁄ D exchange in GIIIA and SmIIIA. The correctly folded or reduced forms of GIIIA and SmIIIA were allowed to exchange protons in deuterated phosphate buffer pH 7.5. The H ⁄ D exchange reaction was quenched by mixing the aliquots with chilled matrix solution (a-cyano-4-hydroxycinnamic acid) prepared in a mixture of acetonitrile ⁄ ethanol ⁄ 0.1% TFA in D 2 O(1:1:1, v ⁄ v ⁄ v). The D-mass was calculated from differences of the masses prior and after the exchange reaction. D-Mass is defined as zero at time zero. The insert represents H ⁄ D exchange of the reduced forms of GIIIA (d) and SmIIIA (j). Table 2. Structure–stability relationships for conotoxins. #, C-terminal amidation; Z and O, pyroglutamate and 4-hydroxyproline, respectively. Conotoxin Sequence Folding Yields Ref x-Conotoxins a MVIIA CKGKGAKCSRLMYDCCTGSCRSGKC# 50% [3] MVIIC CKGKGAPCRKTMYDCCSGSCGRRGKC# 16% MVIID CQGRGASCRKTMYNCCSGSCNRGRC# 28% GVIA CKSOGSSCSOTSYNCCRSCNOOYTKRCY# 50% SVIA CRSSGSOCGVTSICCGRCYRGKCT 50% d-Conotoxins b PVIA EACYAPGTFCGIKPGLCCSEFCLPGVCFG# 3% [6] SVIE EACSSGGTFCGIHPGLCCSEFCFLWCITFID <1% TxVIA WCKQSGEMCNLLDQNCCDGYCIVLVCT 7% GmVIA VKPCRKEGQLCDPIFQNCCRGWNCVLFCV 32% M-superfamily conotoxins c GIIIA RDCCTOOKKCKDRQCKOQRCCA# 29% This study PIIIA ZRLCCGFOKSCRSRQCKOHRCC# 26% SmIIIA ZRCCNGRRGCSSRWCRDHSRCC# 21% RIIIK LOSCCSLNLRLCOVOACKRNOCCT 62% a 0.2 M MOPS, pH 7.3, 0.2 M KCl, 1 mM EDTA, 1 : 2 GSSG ⁄ GSH. b 0.1 M Tris ⁄ HCl, pH 8.7, 1 mM cystamine, 1 mM GSH. c 0.1 M Tris ⁄ HCl, pH 7.5, 1 : 1 GSSG ⁄ GSH. E. Fuller et al. Oxidative folding of conotoxins FEBS Journal 272 (2005) 1727–1738 ª 2005 FEBS 1733 folding mechanism, as two kinetically trapped two- disulfide intermediates significantly decrease the rate for forming the correctly folded species. However, destabilization of these kinetic traps by several single amino acid replacements resulted in a dramatic increase of the rate for forming the correctly folded BPTI [29–31,34]. These mutations were found to destabilize not only the kinetic traps but also other folding inter- mediates and the native protein. The diversity of fold- ing properties of the BPTI mutants can be compared to that of the conotoxins studied here. The slow-rear- rangement folding of GIIIA and RIIIK and the rapid- collapse folding of PIIIA and SmIIIA may also be correlated with their stabilities, as judged by a steady- state accumulation of the correctly folded species. Such correlations between the stability and diverse folding mechanisms were previously shown for other disulfide- containing polypeptides [35–37]. Our attempts to identify underlying structural deter- minants of the distinct folding mechanisms among studied conotoxins met with only limited success. What structural features in the primary amino acid sequences of GIIIA and SmIIIA could account for the slow-rearrangement or rapid-collapse, respectively? The slow rearrangement mechanism observed in GI- IIA might be, at least in part, accounted for by a presence of a hydroxyproline residue in the cis configuration. In GIIIA, Hyp7 and Hyp8 are in the trans and cis conformation, respectively, indicating that the cis–trans isomerization may affect a rate of disulfide bond rearrangement in highly accumulated folding intermediates (‘kinetic traps’). GIIIA folding was significantly affected by the presence of 4 m urea in the folding mixture, suggesting the role of tertiary interactions in stabilizing the native conformation. The rapid-collapse folding of SmIIIA was insensitive to denaturing conditions. The significant effect of high ionic strength on the folding yields suggested that repulsive electrostatic interactions, probably between Arg residues, could negatively influence folding and the stability of the native conformation. Stronger effects of ionic strength on the SmIIIA folding, relat- ive to the other conotoxins is somewhat puzzling, as all conotoxins, including GIIIA, contain a high num- ber of positively charged residues (Table 1). Figure 7 shows neighboring residues next to the disulfide bonds in the correctly folded GIIIA and SmIIIA. At first sight, there are no apparent differences between neigh- boring charged residues next to the disulfide bonds. The lack of obvious differences in electrostatic proper- ties between GIIIA and SmIIIA may suggest that local electrostatic interactions might occur transiently in the early folding of SmIIIA. At present, it remains unclear what other factors could determine formation of the native disulfide bonds in SmIIIA, rather than random pairing of cysteine residues governed by their spacing [38–40]. Our unpublished data indicated that the C-terminal amidation did not affect folding properties of SmIIIA (E. Fuller & G. Bulaj, unpublished data), as previously shown in the case of x-MVIIA [26]. Taken together, our results suggest that local properties are more important in determining folding mechanisms rather than the disulfide framework or conformational flexibility. Fig. 7. Comparison of neighboring residues next to the disulfide bonds in the correctly folded forms of GIIIA and SmIIIA. Notice that for SmIIIA all disulfide bonds are located next to a number of positively charged residues. Disulfide bonds in GIIIA were also found next to the positively charged residues, but the negatively charged Asp2 and Asp12 may partially neutralize the positive charges of the Lys and Arg side chains. The models were generated using PDBVIEWER, and PDB files 1TCG (GIIIA) and 1Q2J (SmIIIA). Cys, yellow; Arg, Lys, blue; Asp, Glu, red. Oxidative folding of conotoxins E. Fuller et al. 1734 FEBS Journal 272 (2005) 1727–1738 ª 2005 FEBS There are several implicit questions related to this work. Is the unprecedented sequence diversity among 100 000 conotoxins translated to equally impressive diversity of folding mechanisms? Are there differences between mechanisms of in vitro and in vivo folding of conotoxins? and if there are such differences, what are the primary determinants of the in vivo folding mech- anism of conotoxins? The results presented here addressed only the in vitro folding properties of cono- toxins, but our finding that differences in the folding kinetics for GIIIA and SmIIIA could be lessened in the presence of PDI is somewhat intriguing. PDI is a key enzyme that promotes oxidative folding of poly- peptides in the endoplasmic reticulum [41–43]. PDI- catalyzed folding of a- and x-conotoxins revealed that the enzyme significantly increased apparent folding rates, but not yields for the correctly folded species [2,26]. Interestingly, we did observe significant effects of PDI on GIIIA folding, similar to those for the a-conotoxin GI precursor [2], but PDI did not change the folding kinetics of SmIIIA (this study) or MVIIA [26]. The observed variations could in part be accoun- ted for by different properties of individual conotoxins as PDI substrates [44–46]. The PDI efficiency could also be influenced by the chemical nature of folding reactions (e.g. rearrangement of intramolecular disulfide bond within a kinetic trap or the formation of an intermolecular mixed disulfide, etc.) that might be rate- limiting in the formation of the correctly folded species. The long-term goal of this research is to understand relationships between in vitro and in vivo folding mechanisms of conotoxins, in particular how folding kinetics, thermodynamics and the distribution of folding intermediates can be affected by the pres- ence of molecular crowding, molecular chaperones and folding catalysts, such as PDI or peptidyl proline isom- erase. This work emphasizes differences in the in vitro folding properties of conotoxins, and their in vivo fold- ing mechanisms remain to be elucidated. Experimental procedures Solid-phase peptide synthesis Peptides were synthesized on methylbenzhydrylamine resin using double couplings and standard N-(9-fluorenyl)meth- oxycarbonyl (Fmoc) chemistry. The peptides were removed from a solid support by treatment with reagent K (TFA ⁄ water ⁄ ethanedithiol ⁄ phenol ⁄ thioanisole; 90 : 5 : 2.5 : 7.5 : 5, v ⁄ v ⁄ v ⁄ v ⁄ v). The cleaved peptides were filtered, precipitated with methyl-tert butyl ether (MTBE), precooled at )20 °C and washed several times with cold MTBE. The reduced peptides were purified by reversed-phase HPLC using a pre- parative C 18 Vydac column (218TP1022) in a linear gradi- ent of acetonitrile (ACN) in 0.1% (v ⁄ v) TFA. The flow rate was 10 mLÆmin )1 , and elution was monitored by UV detection at 210 nm. Identity of each peptide was confirmed by MALDI-TOF MS analysis. The peptides were dried by lyophilization and resuspended in 0.01% (v ⁄ v) TFA in water prior to folding experiments. Table 3 summarizes cleavage time and HPLC gradient conditions for each conotoxin. Oxidative folding Folding reactions were carried out in buffered solutions (0.1 m Tris ⁄ HCl), pH 7.5, containing 1 mm EDTA, GSH and GSSG, and when appropriate sodium chloride or urea. Details of folding experiments were described previously [2,47]. The folding reactions were initiated by adding 20 lL of the reduced peptide [resuspended in 0.01% (v ⁄ v) TFA] to 0.2 mL of the folding solution. The final peptide concen- tration was 20 lm. After an appropriate time, aliquots were withdrawn and the reaction was quenched by acidification with formic acid to 8% final concentration. The reaction mixtures were analyzed by analytical reversed-phase HPLC separations using a Vydac C 18 column (218TP54, 4.6 · 250 mm) in a gradient of ACN, as specified in Table 3. The flow rate was 1 mLÆmin )1 , and the HPLC elu- ent was monitored at 220 nm. The folding yields were calculated from the integrated HPLC peak areas. In the reactions with PDI (Sigma-Aldrich, St. Louis, MO, USA), 2 lm enzyme (final concentration) was added to the folding mixture prior to addition of the reduced peptide. Mass spectrometry Molecular masses were determined by MALDI-TOF on a Bruker Daltonics Omni FLEX spectrometer (Billerica, MA, USA). Samples were dissolved in 0.1% (v ⁄ v) TFA and mixed with matrix (a-cyano-4-hydroxy cinnamic acid) sus- pended in 70% (v ⁄ v) ACN in water containing 0.1% (v ⁄ v) TFA. Table 3. Experimental conditions for cleavage and purification of conotoxins used in the folding experiments. Peptide Cleavage time with reagent K (hours) Preparative HPLC gradient a Analytical HPLC gradient GIIIA 4 5% to 30% in 25 min 5% to 30% in 25 min PIIIA 4.5 5% to 35% in 30 min 5% to 30% in 25 min SmIIIA 8 10% to 50% in 40 min 5% to 30% in 25 min RIIIK 4.5 10% to 40% in 30 min 15% to 45% in 30 min a Linear gradient from the initial X% solvent A to the final Y% sol- vent A mixed with solvent B; solvent A, 0.1% TFA; solvent B, 90% ACN ⁄ 0.1% TFA. E. Fuller et al. Oxidative folding of conotoxins FEBS Journal 272 (2005) 1727–1738 ª 2005 FEBS 1735 Hydrogen/deuterium (H/D) exchange Matrix solution a-cyano-4-hydroxycinnamic acid at 5mgÆmL )1 concentration was prepared in a mixture of ACN ⁄ ethanol ⁄ 0.1% (v ⁄ v) TFA in D 2 O (1 : 1 : 1, v ⁄ v⁄ v; D 2 O as 99.9% atom D; Aldrich). The pH of the matrix mixture was then verified to be less than pH 2.5 using col- orpHast indicator strips (EMD Chemicals, Gibbstown, NJ, USA). Each peptide (2.5 nmol) was dissolved in 25 lL water. Deuterated sodium-phosphate buffer (75 lL), pH 7.0, was added to each reaction tube to commence H ⁄ D exchange. Aliquots of 15 lL were removed from the exchange reaction solution and quenched by mixing (1 : 1, v ⁄ v) with the chilled matrix solution at appropriate time points. Next, 1 nmol samples were taken from the quenched reactions and immediately spotted onto a chilled Scout mass spectrometry chip. Samples were dried for 1 min in vacuo and immediately analyzed in positive reflec- ton mode using Omniflex MALDI-TOF mass spectrometry (Bruker Daltonics). The isotopic peaks were smoothed by centroid fitting, and the average molecular mass of the mass envelope was determined. The average mass shifts of the individual peptides were then calculated using xmass soft- ware (Omniflex). Acknowledgements This work was supported in part by NIH Program Project GM 48677 (to B.M.O.). We thank Drs David P. Goldenberg and Martin Horvath for critical reading of the manuscript and their excellent comments. References 1 Terlau H & Olivera BM (2004) Conus venoms: a rich source of novel ion channel-targeted peptides. Physiol Rev 84, 41–68. 2 Buczek O, Olivera BM & Bulaj G (2004) Propeptide does not act as an intramolecular chaperone but facili- tates protein disulfide isomerase-assisted folding of a conotoxin precursor. Biochemistry 43, 1093–1101. 3 Price-Carter M, Gray WM & Goldenberg DP (1996) Folding of x-conotoxins. 1. Efficient disulfide-coupled folding of mature sequences in vitro. Biochemistry 35, 15537–15546. 4 Zhmak MN, Kasheverov IE, Utkin IN, Tsetlin VI, Vol’pina OM & Ivanov VT (2001) [Efficient synthesis of natural a-conotoxins and their analogs]. Bioorg Khim 27, 83–88. 5 Nielsen JS, Buczek P & Bulaj G (2004) Cosolvent- assisted oxidative folding of a bicyclic a-conotoxin ImI. J Pept Sci 10, 249–256. 6 DeLa Cruz R, Whitby FG, Buczek O & Bulaj G (2003) Detergent-assisted oxidative folding of d -conotoxins. J Pept Res 61, 202–212. 7 Bulaj G, Buczek O, Goodsell I, Jimenez EC, Kranski J, Nielsen JS, Garrett JE & Olivera BM (2003) Efficient oxidative folding of conotoxins and the radiation of venomous cone snails. Proc Natl Acad Sci USA 100 (Suppl. 2), 14562–14568. 8 Lancelin JM, Kohda D, Tate S, Yanagawa Y, Abe T, Satake M & Inagaki F (1991) Tertiary structure of con- otoxin GIIIA in aqueous solution. Biochemistry 30, 6908–6916. 9 Hill JM, Alewood PF & Craik DJ (1996) Three dimen- sional solution structure of l-conotoxin GIIIB, a speci- fic blocker of skeletal muscle sodium channels. Biochemistry 35, 8824–8835. 10 Nielsen KJ, Watson M, Adams DJ, Hammarstro ¨ m AK, Gage PW, Hill JM, Craik DJ, Thomas L, Adams D, Alewood PF & Lewis RJ (2002) Solution structure of l-conotoxin PIIIA, a preferential inhibitor of persistent tetrodotoxin-sensitive sodium channels. J Biol Chem 277, 27247–27255. 11 Keizer DW, West PJ, Lee EF, Yoshikami D, Olivera BM, Bulaj G & Norton RS (2003) Structural basis for tetrodotoxin-resistant sodium channel binding by l-con- otoxin SmIIIA. J Biol Chem 278, 46805–46813. 12 Al-Sabi A, Lennartz D, Ferber M, Gulyas J, Rivier JE, Olivera BM, Carlomagno T & Terlau H (2004) jM-Conotoxin RIIIK, structural and functional novelty in a K + channel antagonist. Biochemistry 43, 8625– 8635. 13 Sato K, Ishida Y, Wakamatsu K, Kato R, Honda H, Ohizumi Y, Nakamura H, Ohya M, Lancelin JM, Kohda D & Inagaki F (1991) Active site l-conotoxin GIIIA, a peptide blocker of muscle sodium channels. J Biol Chem 266, 16989–16991. 14 Becker S, Prusak-Sochaczewski E, Zamponi G, Beck- Sickinger AG, Gordon RD & French RJ (1992) Action of derivatives of l-conotoxin GIIIA on sodium chan- nels. Single amino acid substitutions in the toxin sepa- rately affect association and dissociation rates. Biochemistry 31, 8229–8238. 15 Nakamura M, Niwa Y, Ishida Y, Kohno T, Sato K, Oba Y & Nakamura H (2001) Modification of Arg-13 of l-conotoxin GIIIA with piperidinyl-Arg analogs and their relation to the inhibition of sodium channels. FEBS Lett 503, 107–110. 16 Ferber M, Sporning A, Jeserich G, DeLa Cruz R, Wat- kins M, Olivera BM & Terlau H (2003) A novel Conus peptide ligand for K + channels. J Biol Chem 278, 2177– 2183. 17 Ferber M, Al-Sabi A, Stocker M, Olivera BM & Terlau H (2004) Identification of a mammalian target of jM-conotoxin RIIIK. Toxicon 43, 915–921. 18 Cruz LJ, Gray WR, Olivera BM, Zeikus RD, Kerr L, Yoshikami D & Moczydlowski E (1985) Conus geogra- phus toxins that discriminate between neuronal and muscle sodium channels. J Biol Chem 260, 9280–9288. Oxidative folding of conotoxins E. Fuller et al. 1736 FEBS Journal 272 (2005) 1727–1738 ª 2005 FEBS [...]... mechanism for the diversity of disulfide folding pathways J Biol Chem 275, 8287–8289 36 Chang JY & Li L (2002) Pathway of oxidative folding of a-lactalbumin: a model for illustrating the diversity of disulfide folding pathways Biochemistry 41, 8405–8413 37 Chang JY (2004) Evidence for the underlying cause of diversity of the disulfide folding pathway Biochemistry 43, 4522–4529 38 Zhang RM & Snyder GH (1991)... formation of specific disulfides in synthetic variants of a-conotoxin Biochemistry 30, 11343–11348 39 Price-Carter M, Hull MS & Goldenberg DP (1998) Roles of individual disulfide bonds in the stability and folding of an x-conotoxin Biochemistry 37, 9851–9861 40 Price-Carter M, Bulaj G & Goldenberg DP (2002) Initial disulfide formation steps in the folding of an x-conotoxin Biochemistry 41, 3507–3519 41 Freedman... distribution of folding intermediates Protein Sci 6, 1549–1562 31 Zhang JX & Goldenberg DP (1997) Mutational analysis of the BPTI folding pathway II Effects of aromatic fi leucine substitutions on folding kinetics and thermodynamics Protein Sci 6, 1563–1576 FEBS Journal 272 (2005) 1727–1738 ª 2005 FEBS Oxidative folding of conotoxins 32 Goldenberg DP, Frieden RW, Haack JA & Morrison TB (1989) Mutational analysis... isomerase and assisted protein folding J Biol Chem 272, 29399– 29402 24 Villanueva J, Canals F, Villegas V, Querol E & Aviles FX (2000) Hydrogen exchange monitored by MALDI-TOF mass spectrometry for rapid characterization of the stability and conformation of proteins FEBS Lett 472, 27–33 25 Smith DL, Deng Y & Zhang Z (1997) Probing the noncovalent structure of proteins by amide hydrogen exchange and mass... Protein disulfide isomerase: building bridges in protein folding Trends Biochem Sci 9, 331–336 42 Tu BP & Weissman JS (2004) Oxidative protein folding in eukaryotes: mechanisms and consequences J Cell Biol 164, 341–346 43 Wilkinson B & Gilbert HF (2004) Protein disulfide isomerase Biochim Biophys Acta 1699, 35–44 44 Morjana NA & Gilbert HF (1991) Effect of protein and peptide inhibitors on the activity of. .. cladistic analysis Biochemistry 40, 13201–13208 48 West PJ, Bulaj G, Garrett JE, Olivera BM & Yoshikami D (2002) l-Conotoxin SmIIIA, a potent inhibitor of TTX-resistant sodium channels in amphibian sympathetic and sensory neurons Biochemistry 41, 15388– 15393 49 DudaTF Jr & Palumbi SR (1999) Molecular genetics of ecological diversification: duplication and rapid 1737 Oxidative folding of conotoxins. .. Goldenberg DP (1996) Folding of x -conotoxins 2 Influence of precursor sequences and protein disulfide isomerase Biochemistry 35, 15547–15557 27 Kohno T, Sasaki T, Kobayashi K, Fainzilber M & Sato K (2002) Three-dimensional solution structure of the sodium channel agonist ⁄ antagonist d-conotoxin TxVIA J Biol Chem 277, 36387–36391 28 Volpon L, Lamthanh H, Barbier J, Gilles N, Molgo J, Menez A & Lancelin JM (2004)... analysis of a protein -folding pathway Nature 338, 127–132 33 Bulaj G & Goldenberg DP (2001) Phi-values for BPTI folding intermediates and implications for transition state analysis Nat Struct Biol 8, 326–330 34 Bulaj G & Goldenberg DP (2001) Mutational analysis of hydrogen bonding residues in the BPTI folding pathway J Mol Biol 313, 639–656 35 Chang JY, Li L & Bulychev A (2000) The underlying mechanism... structures of d-conotoxin EVIA from Conus ermineus that selectively acts on vertebrate neuronal Na+ channels J Biol Chem 279, 21356–21366 29 Zhang JX & Goldenberg DP (1993) Amino acid replacement that eliminates kinetic traps in the folding pathway of pancreatic trypsin inhibitor Biochemistry 32, 14075–14081 30 Zhang JX & Goldenberg DP (1997) Mutational analysis of the BPTI folding pathway I Effects of aromatic... of toxin genes of the venomous gastropod Conus Proc Natl Acad Sci USA 96, 6820–6823 50 Duda Jr TF & Palumbi SR (2000) Evolutionary diversification of multigene families: allelic selection of toxins in predatory cone snails Mol Biol Evol 17, 1286–1293 51 Conticello SG, Gilad Y, Avidan N, Ben-Asher E, Levy Z & Fainzilber M (2001) Mechanisms for evolving 1738 E Fuller et al hypervariability: the case of . Oxidative folding of conotoxins sharing an identical disulfide bridging framework Erika Fuller 1 , Brad R. Green 1 ,. present a comparative analysis of the folding properties of four conotoxins sharing an identical pattern of cysteine resi- dues forming three disulfide bridges,