The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Salvador Ventura - One of the best experts on this subject based on the ideXlab platform.
-
Plasticity in the Oxidative Folding Pathway of the High Affinity Nerita Versicolor Carboxypeptidase Inhibitor (NvCI)
Scientific Reports, 2017Co-Authors: Sebastián A. Esperante, Silvia Bronsoms, Francesc X Aviles, Giovanni Covaleda, Sebastián A. Trejo, Salvador VenturaAbstract:Nerita Versicolor carboxypeptidase inhibitor (NvCI) is the strongest inhibitor reported so far for the M14A subfamily of carboxypeptidases. It comprises 53 residues and a protein fold composed of a two-stranded antiparallel β sheet connected by three loops and stabilized by three disulfide bridges. Here we report the Oxidative Folding and reductive unFolding pathways of NvCI. Much debate has gone on whether protein conformational Folding guides disulfide bond formation or instead they are disulfide bonds that favour the arrangement of local or global structural elements. We show here that for NvCI both possibilities apply. Under physiological conditions, this protein folds trough a funnelled pathway involving a network of kinetically connected native-like intermediates, all sharing the disulfide bond connecting the two β-strands. In contrast, under denaturing conditions, the Folding of NvCI is under thermodynamic control and follows a “trial and error” mechanism, in which an initial quasi-stochastic population of intermediates rearrange their disulfide bonds to attain the stable native topology. Despite their striking mechanistic differences, the efficiency of both Folding routes is similar. The present study illustrates thus a surprising plasticity in the Folding of this extremely stable small disulfide-rich inhibitor and provides the basis for its redesign for biomedical applications.
-
the mitochondrial intermembrane space oxireductase mia40 funnels the Oxidative Folding pathway of the cytochrome c oxidase assembly protein cox19
Journal of Biological Chemistry, 2014Co-Authors: Hugo Fraga, Joanjosep Bechserra, Francesc Canals, Gabriel Ortega, Oscar Millet, Salvador VenturaAbstract:Mia40-catalyzed disulfide formation drives the import of many proteins into the mitochondria. Here we characterize the Oxidative Folding of Cox19, a twin CX9C Mia40 substrate. Cox19 oxidation is extremely slow, explaining the persistence of import-competent reduced species in the cytosol. Mia40 accelerates Cox19 Folding through the specific recognition of the third Cys in the second helical CX9C motif and the subsequent oxidation of the inner disulfide bond. This renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. The same intermediate dominates the pathway in the absence of Mia40, and chemical induction of an α-helical structure by trifluoroethanol suffices to accelerate productive Folding and mimic the Mia40 Folding template mechanism. The Mia40 role is to funnel a rough Folding landscape, skipping the accumulation of kinetic traps, providing a rationale for the promiscuity of Mia40.
-
Oxidative Folding in the Mitochondrial Intermembrane Space in Human Health and Disease
International Journal of Molecular Sciences, 2013Co-Authors: Hugo Fraga, Salvador VenturaAbstract:Oxidative Folding in the mitochondrial intermembrane space (IMS) is a key cellular event associated with the Folding and import of a large and still undetermined number of proteins. This process is catalyzed by an oxidoreductase, Mia40 that is able to recognize substrates with apparently little or no homology. Following substrate oxidation, Mia40 is reduced and must be reoxidized by Erv1/Alr1 that consequently transfers the electrons to the mitochondrial respiratory chain. Although our understanding of the physiological relevance of this process is still limited, an increasing number of pathologies are being associated with the impairment of this pathway; especially because Oxidative Folding is fundamental for several of the proteins involved in defense against Oxidative stress. Here we review these aspects and discuss recent findings suggesting that Oxidative Folding in the IMS is modulated by the redox state of the cell.
-
Deciphering the structural basis that guides the Oxidative Folding of leech-derived tryptase inhibitor.
The Journal of biological chemistry, 2009Co-Authors: David Pantoja-uceda, Jorge Santoro, Joan L. Arolas, Francesc X Aviles, Salvador Ventura, Christian Petro SommerhoffAbstract:Protein Folding mechanisms have remained elusive mainly because of the transient nature of intermediates. Leech-derived tryptase inhibitor (LDTI) is a Kazal-type serine proteinase inhibitor that is emerging as an attractive model for Folding studies. It comprises 46 amino acid residues with three disulfide bonds, with one located inside a small triple-stranded antiparallel β-sheet and with two involved in a cystine-stabilized α-helix, a motif that is widely distributed in bioactive peptides. Here, we analyzed the Oxidative Folding and reductive unFolding of LDTI by chromatographic and disulfide analyses of acid-trapped intermediates. It folds and unfolds, respectively, via sequential oxidation and reduction of the cysteine residues that give rise to a few 1- and 2-disulfide intermediates. Species containing two native disulfide bonds predominate during LDTI Folding (IIa and IIc) and unFolding (IIa and IIb). Stop/go Folding experiments demonstrate that only intermediate IIa is productive and oxidizes directly into the native form. The NMR structures of acid-trapped and further isolated IIa, IIb, and IIc reveal global folds similar to that of the native protein, including a native-like canonical inhibitory loop. Enzyme kinetics shows that both IIa and IIc are inhibitory-active, which may substantially reduce proteolysis of LDTI during its Folding process. The results reported show that the kinetics of the Folding reaction is modulated by the specific structural properties of the intermediates and together provide insights into the interdependence of conformational Folding and the assembly of native disulfides during Oxidative Folding.
-
scrambled isomers as key intermediates in the Oxidative Folding of ligand binding module 5 of the low density lipoprotein receptor
Journal of Biological Chemistry, 2008Co-Authors: Xabier Ariasmoreno, Joan L. Arolas, Francesc X Aviles, Javier Sancho, Salvador VenturaAbstract:The ligand binding module five (LA5) of the low density lipoprotein receptor is a small, single-domain protein of 40 residues and three disulfide bonds with a calcium binding motif that is essential for its structure and function. Several mutations in LA5 have been reported to cause familial hypercholesterolemia by impairing a proper Folding of the module. The current study reports the Oxidative Folding and reductive unFolding pathways of wild type and mutant LA5 modules through kinetic and structural analysis of the trapped intermediates. Wild type LA5 Folding involves an initial phase of nonspecific packing where the sequential oxidation of its cysteines gives rise to complex equilibrated populations of intermediates. In the presence of calcium, the attainment of a coordination-competent conformation becomes the rate-limiting step of Folding while binding of the ion funnels both thermodynamically and kinetically the Folding reaction toward the native state. In the absence of calcium, a scrambled isomer (termed Xa) constitutes the global free energy minimum of the Folding process. Xa and the native form are stable, inter-convertible species whose relative populations at equilibrium appear displaced in disease-linked mutants toward the scrambled form. Because stable scrambled isomers such as Xa avoid the exposition of reactive cysteines in misfolded modules, they might constitute a strategy to prevent wrong interactions with other domains during Folding of the receptor. Comparison of the Folding pathways of wild type and mutant LA5 provides the molecular basis to understand how LA modules fold into a functional conformation or upon mutation misfold and lead to disease.
Grzegorz Bulaj - One of the best experts on this subject based on the ideXlab platform.
-
distinct disulfide isomers of μ conotoxins kiiia and kiiib block voltage gated sodium channels
Biochemistry, 2012Co-Authors: Keith K Khoo, Grzegorz Bulaj, Doju Yoshikami, Baldomero M Olivera, Min-min Zhang, Kallol Gupta, Brad R Green, Maren Watkins, P Balaram, Raymond S NortonAbstract:In the preparation of synthetic conotoxins containing multiple disulfide bonds, Oxidative Folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of mu-conotoxin KIIIA, which was predicted originally to have a C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely related mu-conotoxins. The two major isomers of synthetic mu-KIIIA formed during Oxidative Folding were purified and their disulfide connectivities mapped by direct mass spectrometric collision-induced dissociation fragmentation of the disulfide-bonded polypeptides. Our results show that the major Oxidative Folding product adopts a C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of Na(v)1.2 (K-d values of 5 and 230 nM, respectively). The solution structure for mu-KIIIA based on nuclear magnetic resonance data was recalculated with the C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the mu-KIIIA structure calculated with the incorrect C1-C9,C2-C15,C4-C16] disulfide pattern, with an alpha-helix spanning residues 7-12. In addition, the major Folding isomers of mu-KIIIB, an N-terminally extended isoform of mu-KIIIA, identified from its cDNA sequence, were isolated. These Folding products had the same disulfide connectivities as mu-KIIIA, and both blocked Na(v)1.2 (K-d values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic mu-KIIIA and mu-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of mu-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snail's repertoire of active peptides.
-
modulation of conotoxin structure and function is achieved through a multienzyme complex in the venom glands of cone snails
Journal of Biological Chemistry, 2012Co-Authors: Helena Safavihemami, Grzegorz Bulaj, Baldomero M Olivera, Andrew M Steiner, Nicholas A Williamson, Dhana G Gorasia, John A Karas, Joanna Gajewiak, Anthony W PurcellAbstract:The Oxidative Folding of large polypeptides has been investigated in detail; however, comparatively little is known about the enzyme-assisted Folding of small, disulfide-containing peptide substrates. To investigate the concerted effect of multiple enzymes on the Folding of small disulfide-rich peptides, we sequenced and expressed protein-disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase, and immunoglobulin-binding protein (BiP) from Conus venom glands. Conus PDI was shown to catalyze the oxidation and reduction of disulfide bonds in two conotoxins, α-GI and α-ImI. Oxidative Folding rates were further increased in the presence of Conus PPI with the maximum effect observed in the presence of both enzymes. In contrast, Conus BiP was only observed to assist Folding in the presence of microsomes, suggesting that additional co-factors were involved. The identification of a complex between BiP, PDI, and nascent conotoxins further suggests that the Folding and assembly of conotoxins is a highly regulated multienzyme-assisted process. Unexpectedly, all three enzymes contributed to the Folding of the ribbon isomer of α-ImI. Here, we identify this alternative disulfide-linked species in the venom of Conus imperialis, providing the first evidence for the existence of a “non-native” peptide isomer in the venom of cone snails. Thus, ER-resident enzymes act in concert to accelerate the Oxidative Folding of conotoxins and modulate their conformation and function by reconfiguring disulfide connectivities. This study has evaluated the role of a number of ER-resident enzymes in the Folding of conotoxins, providing novel insights into the enzyme-guided assembly of these small, disulfide-rich peptides.
-
reagentless Oxidative Folding of disulfide rich peptides catalyzed by an intramolecular diselenide
Angewandte Chemie, 2012Co-Authors: Andrew M Steiner, Baldomero M Olivera, Kenneth J Woycechowsky, Grzegorz BulajAbstract:In cysteine-rich peptides, diselenides can be used as a proxy for disulfide bridges, since the energetic preference for diselenide bonding over mixed selenium-sulfur bonds simplifies Folding. Herein we report that an intramolecular diselenide bond efficiently catalyzes the Oxidative Folding of selenopeptide analogs of conotoxins, and serves as a reagentless method to substantially accelerate formation of various native disulfide bridging patterns.
-
optimization of Oxidative Folding methods for cysteine rich peptides a study of conotoxins containing three disulfide bridges
Journal of Peptide Science, 2011Co-Authors: Andrew M Steiner, Grzegorz BulajAbstract:The Oxidative Folding of small, cysteine-rich peptides to selectively achieve the native disulfide bond connectivities is critical for discovery and structure-function studies of many bioactive peptides. As the propensity to acquire the native conformation greatly depends on the peptide sequence, numerous empirical oxidation methods are employed. The context-dependent optimization of these methods has thus far precluded a generalized Oxidative Folding protocol, in particular for peptides containing more than two disulfides. Herein, we compare the efficacy of optimized solution-phase and polymer-supported oxidation methods using three disulfide-bridged conotoxins, namely µ-SIIIA, µ-KIIIA and ω-GVIA. The use of diselenide bridges as proxies for disulfide bridges is also evaluated. We propose the ClearOx-assisted oxidation of selenopeptides as a fairly generalized Oxidative Folding protocol.
-
disulfide depleted selenoconopeptides simplified Oxidative Folding of cysteine rich peptides
ACS Medicinal Chemistry Letters, 2010Co-Authors: Tiffany S Han, Aleksandra Walewska, Doju Yoshikami, Baldomero M Olivera, Min-min Zhang, Konkallu Hanumae Gowd, Grzegorz BulajAbstract:Despite the therapeutic promise of disulfide-rich, peptidic natural products, their discovery and structure/function studies have been hampered by inefficient Oxidative Folding methods for their synthesis. Here we report that converting the three disulfide-bridged μ-conopeptide KIIIA into a disulfide-depleted selenoconopeptide (by removal of a noncritical disulfide bridge and substitution of another disulfide bridge with a diselenide bridge) dramatically simplified its Oxidative Folding while preserving the peptide’s ability to block voltage-gated sodium channels. The simplicity of synthesizing disulfide-depleted selenopeptide analogues containing a single disulfide bridge allowed rapid positional scanning at Lys7 of μ-KIIIA, resulting in the identification of K7L as a mutation that improved the peptide’s selectivity in blocking a neuronal (Nav1.2) over a muscle (Nav1.4) subtype of sodium channel. The disulfide-depleted selenopeptide strategy offers regioselective Folding compatible with high-throughput c...
Baldomero M Olivera - One of the best experts on this subject based on the ideXlab platform.
-
distinct disulfide isomers of μ conotoxins kiiia and kiiib block voltage gated sodium channels
Biochemistry, 2012Co-Authors: Keith K Khoo, Grzegorz Bulaj, Doju Yoshikami, Baldomero M Olivera, Min-min Zhang, Kallol Gupta, Brad R Green, Maren Watkins, P Balaram, Raymond S NortonAbstract:In the preparation of synthetic conotoxins containing multiple disulfide bonds, Oxidative Folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of mu-conotoxin KIIIA, which was predicted originally to have a C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely related mu-conotoxins. The two major isomers of synthetic mu-KIIIA formed during Oxidative Folding were purified and their disulfide connectivities mapped by direct mass spectrometric collision-induced dissociation fragmentation of the disulfide-bonded polypeptides. Our results show that the major Oxidative Folding product adopts a C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of Na(v)1.2 (K-d values of 5 and 230 nM, respectively). The solution structure for mu-KIIIA based on nuclear magnetic resonance data was recalculated with the C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the mu-KIIIA structure calculated with the incorrect C1-C9,C2-C15,C4-C16] disulfide pattern, with an alpha-helix spanning residues 7-12. In addition, the major Folding isomers of mu-KIIIB, an N-terminally extended isoform of mu-KIIIA, identified from its cDNA sequence, were isolated. These Folding products had the same disulfide connectivities as mu-KIIIA, and both blocked Na(v)1.2 (K-d values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic mu-KIIIA and mu-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of mu-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snail's repertoire of active peptides.
-
modulation of conotoxin structure and function is achieved through a multienzyme complex in the venom glands of cone snails
Journal of Biological Chemistry, 2012Co-Authors: Helena Safavihemami, Grzegorz Bulaj, Baldomero M Olivera, Andrew M Steiner, Nicholas A Williamson, Dhana G Gorasia, John A Karas, Joanna Gajewiak, Anthony W PurcellAbstract:The Oxidative Folding of large polypeptides has been investigated in detail; however, comparatively little is known about the enzyme-assisted Folding of small, disulfide-containing peptide substrates. To investigate the concerted effect of multiple enzymes on the Folding of small disulfide-rich peptides, we sequenced and expressed protein-disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase, and immunoglobulin-binding protein (BiP) from Conus venom glands. Conus PDI was shown to catalyze the oxidation and reduction of disulfide bonds in two conotoxins, α-GI and α-ImI. Oxidative Folding rates were further increased in the presence of Conus PPI with the maximum effect observed in the presence of both enzymes. In contrast, Conus BiP was only observed to assist Folding in the presence of microsomes, suggesting that additional co-factors were involved. The identification of a complex between BiP, PDI, and nascent conotoxins further suggests that the Folding and assembly of conotoxins is a highly regulated multienzyme-assisted process. Unexpectedly, all three enzymes contributed to the Folding of the ribbon isomer of α-ImI. Here, we identify this alternative disulfide-linked species in the venom of Conus imperialis, providing the first evidence for the existence of a “non-native” peptide isomer in the venom of cone snails. Thus, ER-resident enzymes act in concert to accelerate the Oxidative Folding of conotoxins and modulate their conformation and function by reconfiguring disulfide connectivities. This study has evaluated the role of a number of ER-resident enzymes in the Folding of conotoxins, providing novel insights into the enzyme-guided assembly of these small, disulfide-rich peptides.
-
reagentless Oxidative Folding of disulfide rich peptides catalyzed by an intramolecular diselenide
Angewandte Chemie, 2012Co-Authors: Andrew M Steiner, Baldomero M Olivera, Kenneth J Woycechowsky, Grzegorz BulajAbstract:In cysteine-rich peptides, diselenides can be used as a proxy for disulfide bridges, since the energetic preference for diselenide bonding over mixed selenium-sulfur bonds simplifies Folding. Herein we report that an intramolecular diselenide bond efficiently catalyzes the Oxidative Folding of selenopeptide analogs of conotoxins, and serves as a reagentless method to substantially accelerate formation of various native disulfide bridging patterns.
-
disulfide depleted selenoconopeptides simplified Oxidative Folding of cysteine rich peptides
ACS Medicinal Chemistry Letters, 2010Co-Authors: Tiffany S Han, Aleksandra Walewska, Doju Yoshikami, Baldomero M Olivera, Min-min Zhang, Konkallu Hanumae Gowd, Grzegorz BulajAbstract:Despite the therapeutic promise of disulfide-rich, peptidic natural products, their discovery and structure/function studies have been hampered by inefficient Oxidative Folding methods for their synthesis. Here we report that converting the three disulfide-bridged μ-conopeptide KIIIA into a disulfide-depleted selenoconopeptide (by removal of a noncritical disulfide bridge and substitution of another disulfide bridge with a diselenide bridge) dramatically simplified its Oxidative Folding while preserving the peptide’s ability to block voltage-gated sodium channels. The simplicity of synthesizing disulfide-depleted selenopeptide analogues containing a single disulfide bridge allowed rapid positional scanning at Lys7 of μ-KIIIA, resulting in the identification of K7L as a mutation that improved the peptide’s selectivity in blocking a neuronal (Nav1.2) over a muscle (Nav1.4) subtype of sodium channel. The disulfide-depleted selenopeptide strategy offers regioselective Folding compatible with high-throughput c...
-
identification of conus peptidylprolyl cis trans isomerases ppiases and assessment of their role in the Oxidative Folding of conotoxins
Journal of Biological Chemistry, 2010Co-Authors: Helena Safavihemami, Grzegorz Bulaj, Baldomero M Olivera, Nicholas A Williamson, Anthony W PurcellAbstract:Peptidylprolyl cis-trans isomerases (PPIases) are ubiquitous proteins that catalyze the cis-trans isomerization of prolines. A number of proteins, such as Drosophila rhodopsin and the human immunodeficiency viral protein HIV-1 Gag, have been identified as endogenous substrates for PPIases. However, very little is known about the interaction of PPIases with small, disulfide-rich peptides. Marine cone snails synthesize a wide array of cysteine-rich peptides, called conotoxins, many of which contain one or more prolines or hydroxyprolines. To identify whether PPIase-associated cis-trans isomerization of these residues affects the Oxidative Folding of conotoxins, we identified, sequenced, and expressed three functionally active isoforms of PPIase from the venom gland of Conus novaehollandiae, and we characterized their ability to facilitate Oxidative Folding of conotoxins in vitro. Three conotoxins, namely mu-GIIIA, mu-SIIIA, and omega-MVIIC, derived from two distinct toxin gene families were assayed. Conus PPIase significantly increased the rate of appearance of the native form of mu-GIIIA, a peptide containing three hydroxyprolines. In contrast, the presence of PPIase had no effect on the Folding of mu-SIIIA and omega-MVIIC, peptides containing no or one proline residue, respectively. We further showed that an endoplasmic reticulum-resident PPIase isoform facilitated Folding of mu-GIIIA more efficiently than two cytosolic isoforms. This is the first study to demonstrate PPIase-assisted Folding of conotoxins, small disulfide-rich peptides with unique structural properties.
Anthony W Purcell - One of the best experts on this subject based on the ideXlab platform.
-
modulation of conotoxin structure and function is achieved through a multienzyme complex in the venom glands of cone snails
Journal of Biological Chemistry, 2012Co-Authors: Helena Safavihemami, Grzegorz Bulaj, Baldomero M Olivera, Andrew M Steiner, Nicholas A Williamson, Dhana G Gorasia, John A Karas, Joanna Gajewiak, Anthony W PurcellAbstract:The Oxidative Folding of large polypeptides has been investigated in detail; however, comparatively little is known about the enzyme-assisted Folding of small, disulfide-containing peptide substrates. To investigate the concerted effect of multiple enzymes on the Folding of small disulfide-rich peptides, we sequenced and expressed protein-disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase, and immunoglobulin-binding protein (BiP) from Conus venom glands. Conus PDI was shown to catalyze the oxidation and reduction of disulfide bonds in two conotoxins, α-GI and α-ImI. Oxidative Folding rates were further increased in the presence of Conus PPI with the maximum effect observed in the presence of both enzymes. In contrast, Conus BiP was only observed to assist Folding in the presence of microsomes, suggesting that additional co-factors were involved. The identification of a complex between BiP, PDI, and nascent conotoxins further suggests that the Folding and assembly of conotoxins is a highly regulated multienzyme-assisted process. Unexpectedly, all three enzymes contributed to the Folding of the ribbon isomer of α-ImI. Here, we identify this alternative disulfide-linked species in the venom of Conus imperialis, providing the first evidence for the existence of a “non-native” peptide isomer in the venom of cone snails. Thus, ER-resident enzymes act in concert to accelerate the Oxidative Folding of conotoxins and modulate their conformation and function by reconfiguring disulfide connectivities. This study has evaluated the role of a number of ER-resident enzymes in the Folding of conotoxins, providing novel insights into the enzyme-guided assembly of these small, disulfide-rich peptides.
-
identification of conus peptidylprolyl cis trans isomerases ppiases and assessment of their role in the Oxidative Folding of conotoxins
Journal of Biological Chemistry, 2010Co-Authors: Helena Safavihemami, Grzegorz Bulaj, Baldomero M Olivera, Nicholas A Williamson, Anthony W PurcellAbstract:Peptidylprolyl cis-trans isomerases (PPIases) are ubiquitous proteins that catalyze the cis-trans isomerization of prolines. A number of proteins, such as Drosophila rhodopsin and the human immunodeficiency viral protein HIV-1 Gag, have been identified as endogenous substrates for PPIases. However, very little is known about the interaction of PPIases with small, disulfide-rich peptides. Marine cone snails synthesize a wide array of cysteine-rich peptides, called conotoxins, many of which contain one or more prolines or hydroxyprolines. To identify whether PPIase-associated cis-trans isomerization of these residues affects the Oxidative Folding of conotoxins, we identified, sequenced, and expressed three functionally active isoforms of PPIase from the venom gland of Conus novaehollandiae, and we characterized their ability to facilitate Oxidative Folding of conotoxins in vitro. Three conotoxins, namely mu-GIIIA, mu-SIIIA, and omega-MVIIC, derived from two distinct toxin gene families were assayed. Conus PPIase significantly increased the rate of appearance of the native form of mu-GIIIA, a peptide containing three hydroxyprolines. In contrast, the presence of PPIase had no effect on the Folding of mu-SIIIA and omega-MVIIC, peptides containing no or one proline residue, respectively. We further showed that an endoplasmic reticulum-resident PPIase isoform facilitated Folding of mu-GIIIA more efficiently than two cytosolic isoforms. This is the first study to demonstrate PPIase-assisted Folding of conotoxins, small disulfide-rich peptides with unique structural properties.
-
identification of conus peptidylprolyl cis trans isomerases ppiases and assessment of their role in the Oxidative Folding of conotoxins
Journal of Biological Chemistry, 2010Co-Authors: Helena Safavihemami, Grzegorz Bulaj, Baldomero M Olivera, Nicholas A Williamson, Anthony W PurcellAbstract:Peptidylprolyl cis-trans isomerases (PPIases) are ubiquitous proteins that catalyze the cis-trans isomerization of prolines. A number of proteins, such as Drosophila rhodopsin and the human immunodeficiency viral protein HIV-1 Gag, have been identified as endogenous substrates for PPIases. However, very little is known about the interaction of PPIases with small, disulfide-rich peptides. Marine cone snails synthesize a wide array of cysteine-rich peptides, called conotoxins, many of which contain one or more prolines or hydroxyprolines. To identify whether PPIase-associated cis-trans isomerization of these residues affects the Oxidative Folding of conotoxins, we identified, sequenced, and expressed three functionally active isoforms of PPIase from the venom gland of Conus novaehollandiae, and we characterized their ability to facilitate Oxidative Folding of conotoxins in vitro. Three conotoxins, namely μ-GIIIA, μ-SIIIA, and ω-MVIIC, derived from two distinct toxin gene families were assayed. Conus PPIase significantly increased the rate of appearance of the native form of μ-GIIIA, a peptide containing three hydroxyprolines. In contrast, the presence of PPIase had no effect on the Folding of μ-SIIIA and ω-MVIIC, peptides containing no or one proline residue, respectively. We further showed that an endoplasmic reticulum-resident PPIase isoform facilitated Folding of μ-GIIIA more efficiently than two cytosolic isoforms. This is the first study to demonstrate PPIase-assisted Folding of conotoxins, small disulfide-rich peptides with unique structural properties.
David J. Craik - One of the best experts on this subject based on the ideXlab platform.
-
Cystine Knot Folding in Cyclotides
ChemInform, 2011Co-Authors: Norelle L Daly, Christian W. Gruber, Ulf Göransson, David J. CraikAbstract:Cyclotides are naturally occurring plant-based proteins of approximately 30 amino acids in size that contain a head-to-tail cyclized backbone and a cystine knot motif formed by their three conserved disulfide bonds. Their exceptional stability and unique topology make them valuable frameworks in drug design or protein engineering applications. To facilitate such applications and to explore structure–activity relationships of cyclotides it is useful to be able to chemically synthesize them, a process that is readily achieved via solid phase peptide synthesis followed by Oxidative Folding. This chapter describes what is known about the Oxidative Folding of cyclotides, both in chemical Folding buffers and assisted by a protein disulfide isomerase enzyme isolated from a cyclotide-producing plant. Formation of the cystine knot motif is readily achieved, despite its apparent topological complexity.
-
interlocking disulfides in circular proteins toward efficient Oxidative Folding of cyclotides
Antioxidants & Redox Signaling, 2011Co-Authors: Teshome Leta Aboye, Richard J Clark, David J. Craik, Robert Burman, Marta Bajona Roig, Ulf GöranssonAbstract:Cyclotides are ultrastable plant proteins characterized by the presence of a cyclic amide backbone and three disulfide bonds that form a cystine knot. Because of their extreme stability, there has been significant interest in developing these molecules as a drug design scaffold. For this potential to be realized, efficient methods for the synthesis and Oxidative Folding of cyclotides need to be developed, yet we currently have only a basic understanding of the Folding mechanism and the factors influencing this process. In this study, we determine the major factors influencing Oxidative Folding of the different subfamilies of cyclotides. The Folding of all the cyclotides examined was heavily influenced by the concentration of redox reagents, with the Folding rate and final yield of the native isomer greatly enhanced by high concentrations of oxidized glutathione. Addition of hydrophobic solvents to the buffer also enhanced the Folding rates and appeared to alter the Folding pathway. Significant deamidation and isoaspartate formation were seen when oxidation conditions were conducive to slow Folding. The identification of factors that influence the Folding and degradation pathways of cyclotides will facilitate the development of Folding screens and optimized conditions for producing cyclotides and grafted analogs as stable peptide-based therapeutics.
-
Dissecting the Oxidative Folding of circular cystine knot miniproteins.
Antioxidants & redox signaling, 2009Co-Authors: Sunithi Gunasekera, Richard J Clark, Norelle L Daly, David J. CraikAbstract:Cyclotides are plant proteins with exceptional stability owing to the presence of a cyclic backbone and three disulfide bonds arranged in a cystine knot motif. Accordingly, they have been proposed as templates to stabilize bioactive epitopes in drug-design applications. The two main subfamilies, referred to as the Mobius and bracelet cyclotides, require dramatically different in vitro Folding conditions to achieve the native fold. To determine the underlying elements that influence cyclotide Folding, we examined the in vitro Folding of a suite of hybrid cyclotides based on combination of the Mobius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1. The Folding pathways of the two cyclotide subfamilies were found to be different and influenced by specific residues within intercysteine loops 2 and 6. Two changes in these loops, a substitution in loop 2 and an addition in loop 6, enabled the Folding of a cycloviolacin O1 analogue under conditions in which Folding does not occur in vitro for the native peptide. A key intermediate contains a native-like hairpin structure that appears to be a nucleation locus early in the Folding process. Overall, these mechanistic findings on the Folding of cyclotides are potentially valuable for the design of new drug leads.
-
the structure of a two disulfide intermediate assists in elucidating the Oxidative Folding pathway of a cyclic cystine knot protein
Structure, 2008Co-Authors: Masa Cemažar, Norelle L Daly, Ajinkya Joshi, Alan E Mark, David J. CraikAbstract:We have determined the three-dimensional structure of a two-disulfide intermediate (Cys8-Cys20, Cys14-Cys26) on the Oxidative Folding pathway of the cyclotide MCoTI-II. Cyclotides have a range of bioactivities and, because of their exceptional stability, have been proposed as potential molecular scaffolds for drug design applications. The three-dimensional structure of the stable two-disulfide intermediate shows for the most part identical secondary and tertiary structure to the native state. The only exception is a flexible loop, which is collapsed onto the protein core in the native state, whereas in the intermediate it is more loosely associated with the remainder of the protein. The results suggest that the native fold of the peptide does not represent the free energy minimum in the absence of the Cys1-Cys18 disulfide bridge and that although there is not a large energy barrier, the peptide must transiently adopt an energetically unfavorable state before the final disulfide can form.
-
a novel plant protein disulfide isomerase involved in the Oxidative Folding of cystine knot defense proteins
Journal of Biological Chemistry, 2007Co-Authors: Christian W. Gruber, Richard J Clark, Masa Cemažar, Tomohisa Horibe, Rosemary F Renda, Marilyn A Anderson, David J. CraikAbstract:We have isolated a protein-disulfide isomerase (PDI) from Oldenlandia affinis (OaPDI), a coffee family (Rubiaceae) plant that accumulates knotted circular proteins called cyclotides. The novel plant PDI appears to be involved in the biosynthesis of cyclotides, since it co-expresses and interacts with the cyclotide precursor protein Oak1. OaPDI exhibits similar isomerase activity but greater chaperone activity than human PDI. Since domain c of OaPDI is predicted to have a neutral pI, we conclude that this domain does not have to be acidic in nature for PDI to be a functional chaperone. Its redox potential of -157 ± 4 mV supports a role as a functional oxidoreductase in the plant. The mechanism of enzyme-assisted Folding of plant cyclotides was investigated by comparing the Folding of kalata B1 derivatives in the presence and absence of OaPDI. OaPDI dramatically enhanced the correct Oxidative Folding of kalata B1 at physiological pH. A detailed investigation of Folding intermediates suggested that disulfide isomerization is an important role of the new plant PDI and is an essential step in the production of insecticidal cyclotides.
