Disulfide-rich Peptides Research Articles

Disulfide-Rich Self-Assembling Peptides Based on Aromatic Amino Acid.

Aromatic residues in assembling peptides play a crucial role in driving peptide self-assembly through π-π stacking and hydrophobic interactions. Although various aromatic capping groups have been extensively studied, systematic investigations into the effects of single aromatic amino acids in assembling peptides remain limited. In this study, the influence of aromatic-aromatic interactions on disulfide-rich assembling peptides is systematically investigated by incorporating three different aromatic amino acids. Their folding propensity, self-assembling properties, and rheological behaviors are evaluated. These results indicate that different aromatic-aromatic interactions have a significant effect on self-assembly abilities, as determined by critical aggregation concentration (CAC) measurements. Furthermore, the biocompatibility of these hydrogels is assessed, and their potential for 3D cell culture is explored. The injectable F1-ox hydrogel demonstrate excellent biocompatibility for SHED and NIH3T3 cells and exhibit a porous structure that facilitates nutrient and cellular metabolic waste exchange. This work provides new insights into the molecular design of peptide-based biomaterials, with a focus on the impact of aromatic residues on disulfide-rich assembling peptides.

Development of an Intravenously Stable Disulfide-Rich Peptide for the Treatment of Chemotherapy-Induced Neuropathic Pain.

α-conotoxins (α-Ctxs), a class of disulfide-rich conopetides, are excellent drug leads due to their small size, high selectivity, and potency for specific membrane receptors and ion channels involved in pain transmission. However, their high susceptibility to proteolytic degradation limits their therapeutic potential. In this study, we designed and synthesized a series of conformationally stable analogues of α-Ctx Mr1.1[S4Dap] using various structural optimization strategies. The Mr1.1[S4Dap, C16Pen] analogue maintained potency at human α9α10 nicotinic acetylcholine receptors, with a half-maximal inhibitory concentration (IC50) of 4 nM. It exhibited over a 5-fold increase in serum stability compared to Mr1.1[S4Dap], without disrupting its overall conformation. Furthermore, intravenous application of Mr1.1[S4Dap, C16Pen] showed potent analgesic activity in oxaliplatin-induced cold allodynia, indicating a high potential for drug development. Overall, the results from this study provide valuable insights for optimizing the serum stability of disulfide-rich peptides in future therapeutic applications.

Sec-isoamyl Mercaptan (SIT), a Multi-faceted Disulfide Based Protecting Group for Cysteine Thiol

IntroductionThe successful synthesis of a peptide requires the synchronization of several processes, including the efficient execution of protecting group chemistry. For cysteine (Cys)-peptides, this is more crucial because the trifunctional Cys has a free thiol in its side chain. During synthesis, this free thiol function remains protected with suitable protecting groups and can be removed after synthesis using appropriate methods. Sec-isoamyl mercaptan (SIT) is a versatile disulfide-based protecting group for Cys side chain thiol. The removal of SIT from Cys thiol can be achieved using a mild reducing agent (e. g. DTT). This later promotes efficient disulfide bond formation by oxidation. SIT can also direct/activate the Cys thiol for the chemoselective formation of disulfide bonds by thiol-disulfide interchange.MethodsPeptides were synthesized using solid-phase peptide synthesis techniques. The removal of the SIT group was carried out either in the solid phase or in the solution.ResultsIn the present study, we have shown that SIT can be efficiently removed both in solution and on-resin to facilitate disulfide-bridged peptide synthesis. This was exemplified by two syntheses of an atosiban derivative, where the SIT was removed in solution or in solid-phase. Furthermore, a SIT-based facile one-pot synthesis pathway was devised for disulfide-rich peptides. The strategy was faster and greener as it did not involve using an oxidizer. Conotoxin (two S–S) and linaclotide amide (three S–S) were successfully synthesized by adopting the SIT-based strategy. Finally, a racemization study was carried out for SIT, Trt and StBu-protected Cys-peptides. In all cases, SIT-protected peptides showed lesser racemization than StBu-protected peptides. In some instances (synthesis using DMF), SIT-protected peptides showed less racemization compared to the Trt congeners.ConclusionOverall, the multifaceted use of SIT-protection during the synthesis of disulfide-rich peptides has illustrated its versatility as a Cys thiol protecting group.

δ-Conotoxin Structure Prediction and Analysis through Large-Scale Comparative and Deep Learning Modeling Approaches.

The δ-conotoxins, a class of peptides produced in the venom of cone snails, are of interest due to their ability to inhibit the inactivation of voltage-gated sodium channels causing paralysis and other neurological responses, but difficulties in their isolation and synthesis have made structural characterization challenging. Taking advantage of recent breakthroughs in computational algorithms for structure prediction that have made modeling especially useful when experimental data is sparse, this work uses both the deep-learning-based algorithm AlphaFold and comparative modeling method RosettaCM to model and analyze 18 previously uncharacterized δ-conotoxins derived from piscivorous, vermivorous, and molluscivorous cone snails. The models provide useful insights into the structural aspects of these peptides and suggest features likely to be significant in influencing their binding and different pharmacological activities against their targets, with implications for drug development. Additionally, the described protocol provides a roadmap for the modeling of similar disulfide-rich peptides by these complementary methods.

Single Amino Acid Substitution in Loop1 Switches the Selectivity of α-Conotoxin RegIIA towards the α7 Nicotinic Acetylcholine Receptor.

α-Conotoxins are disulfide-rich peptides obtained from the venom of cone snails, which are considered potential molecular probes and drug leads for nAChR-related disorders. However, low specificity towards different nAChR subtypes restricts the further application of many α-conotoxins. In this work, a series of loop1 amino acid-substituted mutants of α-conotoxin RegIIA were synthesized, whose potency and selectivity were evaluated by an electrophysiological approach. The results showed that loop1 alanine scanning mutants [H5A]RegIIA and [P6A]RegIIA blocked rα7 nAChR with IC50s of 446 nM and 459 nM, respectively, while their inhibition against rα3β2 and rα3β4 subtypes was negligible, indicating the importance of the fifth and sixth amino acid residues for RegIIA's potency and selectivity. Then, second-generation mutants were designed and synthesized, among which the analogues [H5V]RegIIA and [H5S]RegIIA showed significantly improved selectivity and comparable potency towards rα7 nAChR compared with the native RegIIA. Overall, these findings provide deep insights into the structure-activity relationship of RegIIA, as well as revealing a unique perspective for the further modification and optimization of α-conotoxins and other active peptides.

Structure and bioactivity of an insecticidal trans-defensin from assassin bug venom

Disulfide-rich peptides such as defensins play diverse roles in immunity and ion channel modulation, as well as constituting the bioactive components of many animal venoms. We investigated the structure and bioactivity of U-RDTX-Pp19, a peptide previously discovered in venom of the assassin bug Pristhesancus plagipennis. Recombinant Pp19 (rPp19) was found to possess insecticidal activity when injected into Drosophila melanogaster. A bioinformatic search revealed that domains homologous to Pp19 are produced by assassin bugs and diverse other arthropods. rPp19 co-eluted with native Pp19 isolated from P.plagipennis, which we found is more abundant in hemolymph than venom. We solved the three-dimensional structure of rPp19 using 2D 1H NMR spectroscopy, finding that it adopts a disulfide-stabilized structure highly similar to known trans-defensins, with the same cystine connectivity as human α-defensin (I-VI, II-IV, and III-V). The structure of Pp19 is unique among reported structures of arthropod peptides.

Delta-Conotoxin Structure Prediction and Analysis through Large-scale Comparative and Deep Learning Modeling Approaches.

The delta-conotoxins, a class of peptide produced in the venom of cone snails, are of interest due to their ability to inhibit the inactivation of voltage-gated sodium channels causing paralysis and other neurological responses, but difficulties in their isolation and synthesis have made structural characterization challenging. Taking advantage of recent breakthroughs in computational algorithms for structure prediction that have made modeling especially useful when experimental data is sparse, this work uses both the deep-learning based algorithm AlphaFold and comparative modeling method RosettaCM to model and analyze 18 previously uncharacterized delta-conotoxins derived from piscivorous, vermivorous, and molluscivorous cone snails. The models provide useful insights into structural aspects of these peptides and suggest features likely to be significant in influencing their binding and different pharmacological activities against their targets, with implications for drug development. Additionally, the described protocol provides a roadmap for the modeling of similar disulfide-rich peptides by these complementary methods.

Identification and Evolutionary Analysis of the Widely Distributed CAP Superfamily in Spider Venom.

Venom plays a crucial role in the defense and predation of venomous animals. Spiders (Araneae) are among the most successful predators and have a fascinating venom composition. Their venom mainly contains disulfide-rich peptides and large proteins. Here, we analyzed spider venom protein families, utilizing transcriptomic and genomic data, and highlighted their similarities and differences. We show that spiders have specific combinations of toxins for better predation and defense, typically comprising a core toxin expressed alongside several auxiliary toxins. Among them, the CAP superfamily is widely distributed and highly expressed in web-building Araneoidea spiders. Our analysis of evolutionary relationships revealed four subfamilies (subA-subD) of the CAP superfamily that differ in structure and potential functions. CAP proteins are composed of a conserved CAP domain and diverse C-terminal domains. CAP subC shares similar domains with the snake ion channel regulator svCRISP proteins, while CAP subD possesses a sequence similar to that of insect venom allergen 5 (Ag5). Furthermore, we show that gene duplication and selective expression lead to increased expression of CAP subD, making it a core member of the CAP superfamily. This study sheds light on the functional diversity of CAP subfamilies and their evolutionary history, which has important implications for fully understanding the composition of spider venom proteins and the core toxin components of web-building spiders.

Proximity Labelling Confirms the Involvement of Papain-Like Cysteine Proteases and Chaperones in Cyclotide Biosynthesis

Cyclotides are disulfide-rich insecticidal peptides from plants that harbour a cyclic cystine knot motif that imparts them with high stability. Like most ribosomally encoded peptides, cyclotides are produced as precursor proteins that are subsequently processed to maturity through proteolytic activity. Although the final maturation of the prototypic cyclotide kalata B1 (kB1) is known to involve asparaginyl endopeptidases, the role of the in planta folding machinery is less well characterized, as is also the case for in planta N-terminal processing enzyme(s). Here, we used proximity labelling to identify proteins involved in the biosynthesis of kB1 in both a cyclotide-bearing plant species, Petunia × hybrida [Regel], and in Nicotiana benthamiana [Domin] that does not naturally produce cyclotides. Together, several ER resident chaperones, protein disulfide isomerases, a papain-like cysteine protease, and an asparaginyl endopeptidase were identified in the kB1 interactome. We found that overexpression of the identified papain-like cysteine protease resulted in an improvement in the yield of cyclic kB1, while this was not the case for the overexpression of protein disulfide isomerases, despite their interaction being verified using bimolecular fluorescence complementation. Together, these results provide a significant advance in our understanding of the role of auxiliary biosynthetic elements for heterologous cyclotide production in plants.

From genome to proteome: Comprehensive identification of venom toxins from the Chinese funnel-web spider (Macrothelidae: Macrothele yani)

Macrothelidae is a family of mygalomorph spiders containing the extant genera Macrothele and Vacrothele. China is an important center of diversity for Macrothele with 65 % of the known species occurring there. Previous work on Macrothele was able to uncover several important toxin compounds including Raventoxin which may have applications in biomedicine and agricultural chemistry. Despite the importance of Macrothele spiders, high-quality reference genomes are still lacking, which hinders our understanding and application of the toxin compounds. In this study, we assembled the genome of the Macrothele yani to help fill gaps in our understanding of toxin biology in this lineage of spiders to encourage the future study and applications of these compounds. The final assembled genome was 6.79 Gb in total length, had a contig N50 of 21.44 Mb, and scaffold N50 of 156.16 Mb. Hi-C scaffolding assigned 98.19 % of the genome to 46 pseudo-chromosomes with a BUSCO score of 95.7 % for the core eukaryotic gene set. The assembled genome was found to contain 75.62 % repetitive DNA and a total of 39,687 protein-coding genes were annotated making it the spider genome with highest number of genes. Through integrated analysis of venom gland transcriptomics and venom proteomics, a total of 194 venom toxins were identified, including 38 disulfide-rich peptide neurotoxins, among which 12 were ICK knottin peptides. In summary, we present the first high-quality genome assembly at the chromosome level for any Macrothelidae spider, filling an important gap in our knowledge of these spiders. Such high-quality genomic data will be invaluable as a reference in resolving Araneae spider phylogenies and in screening different spider species for novel compounds applicable to numerous medical and agricultural applications.

Structural analysis of a U-superfamily conotoxin containing a mini-granulin fold: Insights into key features that distinguish between the ICK and granulin folds

We are entering an exciting time in structural biology where artificial intelligence can be used to predict protein structures with greater accuracy than ever before. Extending this level of accuracy to the predictions of disulfide-rich peptide structures is likely to be more challenging, at least in the short term, given the tight packing of cysteine residues and the numerous ways that the disulfide bonds can potentially be linked. It has been previously shown in many cases that several disulfide bond connectivities can be accommodated by a single set of NMR-derived structural data without significant violations. Disulfide-rich peptides are prevalent throughout nature, and arguably the most well-known are those present in venoms from organisms such as cone snails. Here we have determined the first three-dimensional structure and disulfide connectivity of a U-superfamily cone snail venom peptide, TxVIIB. TxVIIB has a VI/VII cysteine framework that is generally associated with an inhibitor cystine knot (ICK) fold, however AlphaFold predicted that the peptide adopts a mini-granulin fold with a granulin disulfide connectivity. Our experimental studies using NMR spectroscopy and orthogonal protection of cysteine residues indicate that TxVIIB indeed adopts a mini-granulin fold but with the ICK disulfide connectivity. Our findings provide structural insight into the underlying features that govern formation of the mini-granulin fold rather than the ICK fold and will provide fundamental information for prediction algorithms, as the subtle complexity of disulfide isomers may be not adequately addressed by the current prediction algorithms.

Scanning mutagenesis identifies residues that improve the long-term stability and insecticidal activity of cyclotide kalata B1

Cyclotides are plant-derived disulfide-rich cyclic peptides that have a natural function in plant defence and potential for use as agricultural pesticides. Due to their highly constrained topology, they are highly resistant to thermal, chemical or enzymatic degradation. However, the stability of cyclotides at alkaline pH for incubation times of longer than a few days is poorly studied, but important since these conditions could be encountered in the environment, during storage or field application as insecticides. In this study, kalata B1 (kB1), the prototypical cyclotide, was engineered to improve its long-term stability and retain its insecticidal activity via point mutations. We found that substituting either Asn29 or Gly1 to lysine or leucine increased the stability of kB1 by two-fold when incubated in an alkaline buffer (pH 9.0) for seven days, while retaining its insecticidal activity. In addition, when Gly1 was replaced with lysine or leucine, the mutants could be cyclised using an asparaginyl endopeptidase, in vitro with a yield of ∼90% within 5 min. These results demonstrate the potential to manufacture kB1 mutants with increased stability and insecticidal activity recombinantly or in planta. Overall, the discovery of mutants of kB1 that have enhanced stability could be useful in leading to longer term activity in the field as bioinsecticides.

Omniligase-1-Mediated Phage-Peptide Library Modification and Insulin Engineering.

Chemical and enzymatic modifications of peptide-displayed libraries have been successfully employed to expand the phage display library. However, the requirement of specific epitopes and scaffolds has limited the scope of protein engineering using phage display. In this study, we present a novel approach utilizing omniligase-1-mediated selective and specific ligation on the phage pIII protein, offering a high conversion rate and compatibility with commercially available phage libraries. We applied this method to perform high-throughput engineering of insulin analogues with randomized B chain C-terminal regions. Insulin analogues with different B chain C-terminal segments were selected and exhibited biological activity equivalent to that of human insulin. Molecular dynamics studies of insulin analogues revealed a novel interaction between the insulin B27 residue and insulin receptor L1 domain. In summary, our findings highlight the potential of omniligase-1-mediated phage display in the development and screening of disulfide-rich peptides and proteins. This approach holds promise for the creation of novel insulin analogues with enhanced therapeutic properties and exhibits potential for the development of other therapeutic compounds.

Chemical synthesis of grafted cyclotides using a “plug and play” approach

‘Plug and play’ grafting – a simple and convenient modular synthesis method to access complex cyclotide grafts and generate potent GPCR ligands is described.

De Novo Discovery of Cysteine Frameworks for Developing Multicyclic Peptide Libraries for Ligand Discovery.

Conserved cysteine frameworks are essential components of disulfide-rich peptides (DRPs), which dominantly define the structural diversity of both naturally occurring and de novo-designed DRPs. However, there are only very limited numbers of conserved cysteine frameworks, and general methods enabling de novo discovery of cysteine frameworks with robust foldability are still not available. Here, we devised a "touchstone"-based strategy that relies on chasing oxidative foldability between two individual disulfide-rich folds on the phage surface to discover new cysteine frameworks from random sequences. Unique cysteine frameworks with a high degree of compatibility with phage display systems and broad sequence tolerance were successfully identified, which were subsequently exploited for the development of multicyclic DRP libraries, enabling the rapid discovery of new peptide ligands with low-nanomolar and picomolar binding affinity. This study provides an unprecedented method for exploring and exploiting the sequence and structure space of DRPs that is not readily accessible by existing strategies, holding the potential to revolutionize the study of DRPs and significantly advance the design and discovery of multicyclic peptide ligands and drugs.

Basic Residues at Position 11 of α-Conotoxin LvIA Influence Subtype Selectivity between α3β2 and α3β4 Nicotinic Receptors via an Electrostatic Mechanism.

Understanding the determinants of α-conotoxin (α-CTX) selectivity for different nicotinic acetylcholine receptor (nAChR) subtypes is a prerequisite for the design of tool compounds to study nAChRs. However, selectivity optimization of these small, disulfide-rich peptides is difficult not only because of an absence of α-CTX/nAChR co-structures but also because it is challenging to predict how a mutation to an α-CTX will alter its potency and selectivity. As a prototypical system to investigate selectivity, we employed the α-CTX LvIA that is 25-fold selective for the α3β2 nAChR over the related α3β4 nAChR subtype, which is a target for nicotine addiction. Using two-electrode voltage clamp electrophysiology, we identified LvIA[D11R] that is 2-fold selective for the α3β4 nAChR, reversing the subtype preference. This effect is specifically due to the change in charge and not shape of LvIA[D11R], as substitution of D11 with citrulline retains selectivity for the α3β2 nAChR. Furthermore, LvIA[D11K] shows a stronger reversal, with 4-fold selectivity for the α3β4 nAChR. Motivated by these findings, using site-directed mutagenesis, we found that β2[K79A] (I79 on β4), but not β2[K78A] (N78 on β4), largely restores the potency of basic mutants at position 11. Finally, to understand the structural basis of this effect, we used AlphaFold2 to generate models of LvIA in complex with both nAChR subtypes. Both models confirm the plausibility of an electrostatic mechanism to explain the data and also reproduce a broad range of potency and selectivity structure-activity relationships for LvIA mutants, as measured using free energy perturbation simulations. Our work highlights how electrostatic interactions can drive α-CTX selectivity and may serve as a strategy for optimizing the selectivity of LvIA and other α-CTXs.

Development of Melanocortin 4 Receptor Agonists by Exploiting Animal-Derived Macrocyclic, Disulfide-Rich Peptide Scaffolds.

G protein-coupled receptors are among the most widely studied classes of drug targets. A major challenge in this field is to develop ligands that will selectively modulate a single receptor subtype to overcome the disadvantages of undesired "off target" effects caused by lack of target and thus signaling specificity. In the current study, we explored ligand design for the melanocortin 4 receptor (MC4R) since it is an attractive target for developing antiobesity drugs. Endogenously, the receptor is activated by peptide ligands, i.e., three melanocyte-stimulating hormones (α-MSH, β-MSH, and γ-MSH) and by adrenocorticotropic hormone. Therefore, we utilized a peptide drug design approach, utilizing "molecular grafting" of pharmacophore peptide sequence motifs onto a stable nature-derived peptide scaffold. Specifically, protegrin-4-like-peptide-1 (Pr4LP1) and arenicin-1-like-peptide-1 (Ar3LP1) fully activated MC4R in a functional cAMP assay with potencies of 3.7 and 1.0 nM, respectively. In a nanoluciferase complementation assay with less signal amplification, the designed peptides fully recruited mini-Gs with subnanomolar and nanomolar potencies. Interestingly, these novel peptide MC4R ligands recruited β-arrestin-2 with ∼2-fold greater efficacies and ∼20-fold increased potencies as compared to the endogenous α-MSH. The peptides were inactive at related MC1R and MC3R in a cAMP accumulation assay. These findings highlight the applicability of animal-derived disulfide-rich scaffolds to design pathway and subtype selective MC4R pharmacological probes. In the future, this approach could be exploited to develop functionally selective ligands that could offer safer and more effective obesity drugs.

Transcriptome sequencing of wolf spider Lycosa sp. (Araneae: Lycosidae) venom glands provides insights into the evolution and diversity of disulfide-rich toxins

Wolf spiders in the genus Lycosa are important pest predators in agroforestry ecosystems, capable of feeding on a wide range of pests through the use of complex venom which can to quickly immobilize and kill prey. Because of these characteristics the toxins in wolf spiders venom may prove to be natural sources for novel drug development and biopesticides. To better understand the toxins in Lycosa venom we sequenced the transcriptome from venom glands from an undescribed species of Lycosa and comparatively analyzed the data using known protein motifs. A series of 19 disulfide-rich peptide (DRP) toxin sequences were identified and categorized into seven groups based on the number and arrangement of cysteine residues. Notably, we identified three peptide sequences with low identity to any known toxin, which may be toxin peptides specific to this species of Lycosa. In addition, to further understand the evolutionary relationships of disulfide-rich peptide toxins in spider venom, we constructed phylogenetic trees of DRP toxins from three spiders species and found that the Lycosa sp. DRPs are comparatively diverse with previous research results. This study reveals the toxin diversity of wolf spiders (Lycosa sp.) at the transcriptomic level and provides initial insights into the evolution of DRP toxins in spiders, enriching our knowledge of toxin diversity and providing new compounds for functional studies.

Structural and functional characterisation of Tst2, a novel TRPV1 inhibitory peptide from the Australian sea anemone Telmatactis stephensoni

Sea anemone venoms are complex mixtures of biologically active compounds, including disulfide-rich peptides, some of which have found applications as research tools, and others as therapeutic leads. Our recent transcriptomic and proteomic studies of the Australian sea anemone Telmatactis stephensoni identified a transcript for a peptide designated Tst2. Tst2 is a 38-residue peptide showing sequence similarity to peptide toxins known to interact with a range of ion channels (NaV, TRPV1, KV and CaV). Recombinant Tst2 (rTst2, which contains an additional Gly at the N-terminus) was produced by periplasmic expression in Escherichia coli, enabling the production of both unlabelled and uniformly 13C,15N–labelled peptide for functional assays and structural studies. The LC-MS profile of the recombinant Tst2 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds from its six cysteine residues. The solution structure of rTst2 was determined using multidimensional NMR spectroscopy and revealed that rTst2 adopts an inhibitor cystine knot (ICK) structure. rTst2 was screened using various functional assays, including patch–clamp electrophysiological and cytotoxicity assays. rTst2 was inactive against voltage-gated sodium channels (NaV) and the human voltage-gated proton (hHv1) channel. rTst2 also did not possess cytotoxic activity when assessed against Drosophila melanogaster flies. However, the recombinant peptide at 100 nM showed >50% inhibition of the transient receptor potential subfamily V member 1 (TRPV1) and slight (∼10%) inhibition of transient receptor potential subfamily A member 1 (TRPA1). Tst2 is thus a novel ICK inhibitor of the TRPV1 channel.

Structural Conformation and Activity of Spider-Derived Inhibitory Cystine Knot Peptide Pn3a Are Modulated by pH.

Numerous spider venom-derived gating modifier toxins exhibit conformational heterogeneity during purification by reversed-phase high-performance liquid chromatography (RP-HPLC). This conformational exchange is especially peculiar for peptides containing an inhibitor cystine knot motif, which confers excellent structural stability under conditions that are not conducive to disulfide shuffling. This phenomenon is often attributed to proline cis/trans isomerization but has also been observed in peptides that do not contain a proline residue. Pn3a is one such peptide forming two chromatographically distinguishable peaks that readily interconvert following the purification of either conformer. The nature of this exchange was previously uncharacterized due to the fast rate of conversion in solution, making isolation of the conformers impossible. In the present study, an N-terminal modification of Pn3a enabled the isolation of the individual conformers, allowing activity assays to be conducted on the individual conformers using electrophysiology. The conformers were analyzed separately by nuclear magnetic resonance spectroscopy (NMR) to study their structural differences. RP-HPLC and NMR were used to study the mechanism of exchange. The later-eluting conformer was the active conformer with a rigid structure that corresponds to the published structure of Pn3a, while NMR analysis revealed the earlier-eluting conformer to be inactive and disordered. The exchange was found to be pH-dependent, arising in acidic solutions, possibly due to reversible disruption and formation of intramolecular salt bridges. This study reveals the nature of non-proline conformational exchange observed in Pn3a and possibly other disulfide-rich peptides, highlighting that the structure and activity of some disulfide-stabilized peptides can be dramatically susceptible to disruption.