Novel Scorpion Toxin ω-Buthitoxin-Hf1a Selectively Inhibits Calcium Influx via CaV3.3 and CaV3.2 and Alleviates Allodynia in a Mouse Model of Acute Postsurgical Pain.

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 µM) and CaV3.3 (IC50 = 0.49 µM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.

Noncovalent Peptide Stapling Using Alpha-Methyl-l-Phenylalanine for α-Helical Peptidomimetics.

Peptides and peptidomimetics are attractive drug candidates because of their high target specificity and low-toxicity profiles. Developing peptidomimetics using hydrocarbon (HC)-stapling or other stapling strategies has gained momentum because of their high stability and resistance to proteases; however, they have limitations. Here, we take advantage of the α-methyl group and an aromatic phenyl ring in a unique unnatural amino acid, α-methyl-l-phenylalanine (αF), and propose a novel, noncovalent stapling strategy to stabilize peptides. We utilized this strategy to create an α-helical B-chain mimetic of a complex insulin-like peptide, human relaxin-3 (H3 relaxin). Our comprehensive data set (in vitro, ex vivo, and in vivo) confirmed that the new high-yielding B-chain mimetic, H3B10-27(13/17αF), is remarkably stable in serum and fully mimics the biological function of H3 relaxin. H3B10-27(13/17αF) is an excellent scaffold for further development as a drug lead and an important tool to decipher the physiological functions of the neuropeptide G protein-coupled receptor, RXFP3.

Synthetic hookworm-derived peptides are potent modulators of primary human immune cell function that protect against experimental colitis in vivo.

The prevalence of autoimmune diseases is on the rise globally. Currently, autoimmunity presents in over 100 different forms and affects around 9% of the world's population. Current treatments available for autoimmune diseases are inadequate, expensive, and tend to focus on symptom management rather than cure. Clinical trials have shown that live helminthic therapy can decrease chronic inflammation associated with inflammatory bowel disease and other gastrointestinal autoimmune inflammatory conditions. As an alternative and better controlled approach to live infection, we have identified and characterized two peptides, Acan1 and Nak1, from the excretory/secretory component of parasitic hookworms for their therapeutic activity on experimental colitis. We synthesized Acan1 and Nak1 peptides from the Ancylostoma caninum and Necator americanus hookworms and assessed their structures and protective properties in human cell-based assays and in a mouse model of acute colitis. Acan1 and Nak1 displayed anticolitic properties via significantly reducing weight loss and colon atrophy, edema, ulceration, and necrosis in 2,4,6-trinitrobenzene sulfonic acid-exposed mice. These hookworm peptides prevented mucosal loss of goblet cells and preserved intestinal architecture. Acan1 upregulated genes responsible for the repair and restitution of ulcerated epithelium, whereas Nak1 downregulated genes responsible for epithelial cell migration and apoptotic cell signaling within the colon. These peptides were nontoxic and displayed key immunomodulatory functions in human peripheral blood mononuclear cells by suppressing CD4+ T cell proliferation and inhibiting IL-2 and TNF production. We conclude that Acan1 and Nak1 warrant further development as therapeutics for the treatment of autoimmunity, particularly gastrointestinal inflammatory conditions.

Engineering Peptide Inhibitors of the HFE-Transferrin Receptor 1 Complex.

The protein HFE (homeostatic iron regulator) is a key regulator of iron metabolism, and mutations in HFE underlie the most frequent form of hereditary haemochromatosis (HH-type I). Studies have shown that HFE interacts with transferrin receptor 1 (TFR1), a homodimeric type II transmembrane glycoprotein that is responsible for the cellular uptake of iron via iron-loaded transferrin (holo-transferrin) binding. It has been hypothesised that the HFE/TFR1 interaction serves as a sensor to the level of iron-loaded transferrin in circulation by means of a competition mechanism between HFE and iron-loaded transferrin association with TFR1. To investigate this, a series of peptides based on the helical binding interface between HFE and TFR1 were generated and shown to significantly interfere with the HFE/TFR1 interaction in an in vitro proximity ligation assay. The helical conformation of one of these peptides, corresponding to the α1 and α2 helices of HFE, was stabilised by the introduction of sidechain lactam "staples", but this did not result in an increase in the ability of the peptide to disrupt the HFE/TFR1 interaction. These peptides inhibitors of the protein-protein interaction between HFE and TFR1 are potentially useful tools for the analysis of the functional role of HFE in the regulation of hepcidin expression.

The genetic origin of evolidine, the first cyclopeptide discovered in plants, and related orbitides.

Cyclic peptides are reported to have antibacterial, antifungal, and other bioactivities. Orbitides are a class of cyclic peptides that are small, head-to-tail cyclized, composed of proteinogenic amino acids and lack disulfide bonds; they are also known in several genera of the plant family Rutaceae. Melicope xanthoxyloides is the Australian rain forest tree of the Rutaceae family in which evolidine, the first plant cyclic peptide, was discovered. Evolidine (cyclo-SFLPVNL) has subsequently been all but forgotten in the academic literature, so to redress this we used tandem MS and de novo transcriptomics to rediscover evolidine and decipher its biosynthetic origin from a short precursor just 48 residues in length. We also identified another six M. xanthoxyloides orbitides using the same techniques. These peptides have atypically diverse C termini consisting of residues not recognized by either of the known proteases plants use to macrocyclize peptides, suggesting new cyclizing enzymes await discovery. We examined the structure of two of the novel orbitides by NMR, finding one had a definable structure, whereas the other did not. Mining RNA-seq and whole genome sequencing data from other species of the Rutaceae family revealed that a large and diverse family of peptides is encoded by similar sequences across the family and demonstrates how powerful de novo transcriptomics can be at accelerating the discovery of new peptide families.

Structural Characterization of the PawL-Derived Peptide Family, an Ancient Subfamily of Orbitides.

Plants are an excellent source of bioactive peptides, often with disulfide bonds and/or a cyclic backbone. While focus has predominantly been directed at disulfide-rich peptides, a large family of small, cyclic plant peptides lacking disulfide bonds, known as orbitides, has been relatively ignored. A recently discovered subfamily of orbitides is the PawL-derived peptides (PLPs), produced during the maturation of precursors for seed storage albumins. Although their evolutionary origins have been dated, in-depth exploration of the family's structural characteristics and potential bioactivities remains to be conducted. Here we present an extensive and systematic characterization of the PLP family. Nine PLPs were chosen and prepared by solid phase peptide synthesis. Their structural features were studied using solution NMR spectroscopy, and seven were found to possess regions of backbone order. Ordered regions consist of ß-turns, with some PLPs adopting two well-defined ß-turns within sequences as short as seven residues, which are largely the result of side chain interactions. Our data highlight that the sequence diversity within this family results in equally diverse structures. None of these nine PLPs showed antibacterial or antifungal activity.

Barrettides: A Peptide Family Specifically Produced by the Deep-Sea Sponge Geodia barretti.

Natural product discovery by isolation and structure elucidation is a laborious task often requiring ample quantities of biological starting material and frequently resulting in the rediscovery of previously known compounds. However, peptides are a compound class amenable to an alternative genomic, transcriptomic, and in silico discovery route by similarity searches of known peptide sequences against sequencing data. Based on the sequences of barrettides A and B, we identified five new barrettide sequences (barrettides C-G) predicted from the North Atlantic deep-sea demosponge Geodia barretti (Geodiidae). We synthesized, folded, and investigated one of the newly described barrettides, barrettide C (NVVPCFCVEDETSGAKTCIPDNCDASRGTNP, disulfide connectivity I-IV, II-III). Co-elution experiments of synthetic and sponge-derived barrettide C confirmed its native conformation. NMR spectroscopy and the anti-biofouling activity on larval settlement of the bay barnacle Amphibalanus improvisus (IC50 0.64 µM) show that barrettide C is highly similar to barrettides A and B in both structure and function. Several lines of evidence suggest that barrettides are produced by the sponge itself and not one of its microbial symbionts.

Chemical Synthesis and NMR Solution Structure of Conotoxin GXIA from Conus geographus.

Conotoxins are disulfide-rich peptides found in the venom of cone snails. Due to their exquisite potency and high selectivity for a wide range of voltage and ligand gated ion channels they are attractive drug leads in neuropharmacology. Recently, cone snails were found to have the capability to rapidly switch between venom types with different proteome profiles in response to predatory or defensive stimuli. A novel conotoxin, GXIA (original name G117), belonging to the I3-subfamily was identified as the major component of the predatory venom of piscivorous Conus geographus. Using 2D solution NMR spectroscopy techniques, we resolved the 3D structure for GXIA, the first structure reported for the I3-subfamily and framework XI family. The 32 amino acid peptide is comprised of eight cysteine residues with the resultant disulfide connectivity forming an ICK+1 motif. With a triple stranded ß-sheet, the GXIA backbone shows striking similarity to several tarantula toxins targeting the voltage sensor of voltage gated potassium and sodium channels. Supported by an amphipathic surface, the structural evidence suggests that GXIA is able to embed in the membrane and bind to the voltage sensor domain of a putative ion channel target.

Investigation of Receptor Heteromers Using NanoBRET Ligand Binding.

Receptor heteromerization is the formation of a complex involving at least two different receptors with pharmacology that is distinct from that exhibited by its constituent receptor units. Detection of these complexes and monitoring their pharmacology is crucial for understanding how receptors function. The Receptor-Heteromer Investigation Technology (Receptor-HIT) utilizes ligand-dependent modulation of interactions between receptors and specific biomolecules for the detection and profiling of heteromer complexes. Previously, the interacting biomolecules used in Receptor-HIT assays have been intracellular proteins, however in this study we have for the first time used bioluminescence resonance energy transfer (BRET) with fluorescently-labeled ligands to investigate heteromerization of receptors on the cell surface. Using the Receptor-HIT ligand binding assay with NanoBRET, we have successfully investigated heteromers between the angiotensin II type 1 (AT1) receptor and the ß2 adrenergic receptor (AT1-ß2AR heteromer), as well as between the AT1 and angiotensin II type 2 receptor (AT1-AT2 heteromer).

Recifin A, Initial Example of the Tyr-Lock Peptide Structural Family, Is a Selective Allosteric Inhibitor of Tyrosyl-DNA Phosphodiesterase I.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a molecular target for the sensitization of cancer cells to the FDA-approved topoisomerase inhibitors topotecan and irinotecan. High-throughput screening of natural product extract and fraction libraries for inhibitors of TDP1 activity resulted in the discovery of a new class of knotted cyclic peptides from the marine sponge Axinella sp. Bioassay-guided fractionation of the source extract resulted in the isolation of the active component which was determined to be an unprecedented 42-residue cysteine-rich peptide named recifin A. The native NMR structure revealed a novel fold comprising a four strand antiparallel ß-sheet and two helical turns stabilized by a complex disulfide bond network that creates an embedded ring around one of the strands. The resulting structure, which we have termed the Tyr-lock peptide family, is stabilized by a tyrosine residue locked into three-dimensional space. Recifin A inhibited the cleavage of phosphodiester bonds by TDP1 in a FRET assay with an IC50 of 190 nM. Enzyme kinetics studies revealed that recifin A can specifically modulate the enzymatic activity of full-length TDP1 while not affecting the activity of a truncated catalytic domain of TDP1 lacking the N-terminal regulatory domain (Δ1-147), suggesting an allosteric binding site for recifin A on the regulatory domain of TDP1. Recifin A represents both the first of a unique structural class of knotted disulfide-rich peptides and defines a previously unseen mechanism of TDP1 inhibition that could be productively exploited for potential anticancer applications.

Defining the Familial Fold of the Vicilin-Buried Peptide Family.

Plants and their seeds have been shown to be a rich source of cystine-stabilized peptides. Recently a new family of plant seed peptides whose sequences are buried within precursors for seed storage vicilins was identified. Members of this Vicilin-Buried Peptide (VBP) family are found in distantly related plant species including the monocot date palm, as well as dicotyledonous species like pumpkin and sesame. Genetic evidence for their widespread occurrence indicates that they are of ancient origin. Limited structural studies have been conducted on VBP family members, but two members have been shown to adopt a helical hairpin fold. We present an extensive characterization of VBPs using solution NMR spectroscopy, to better understand their structural features. Four peptides were produced by solid phase peptide synthesis and shown to favor a helix-loop-helix hairpin fold, as a result of the I-IV/II-III ladderlike connectivity of their disulfide bonds. Interhelical interactions, including hydrophobic contacts and salt bridges, are critical for the fold stability and control the angle at which the antiparallel α-helices interface. Activities reported for VBPs include trypsin inhibitory activity and inhibition of ribosomal function; however, their diverse structural features despite a common fold suggest that additional bioactivities yet to be revealed are likely.

Insights into the Interaction of LVV-Hemorphin-7 with Angiotensin II Type 1 Receptor.

Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the positive modulation of the angiotensin II (AngII) type 1 receptor (AT1R) by LVV-hemorphin-7 (LVV-H7) in human embryonic kidney (HEK293) cells. Here, we examined the molecular binding behavior of LVV-H7 on AT1R and its effect on AngII binding using a nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay in HEK293FT cells, as well as molecular docking and molecular dynamics (MD) studies. Saturation and real-time kinetics supported the positive effect of LVV-H7 on the binding of AngII. While the competitive antagonist olmesartan competed with AngII binding, LVV-H7 slightly, but significantly, decreased AngII's kD by 2.6 fold with no effect on its Bmax. Molecular docking and MD simulations indicated that the binding of LVV-H7 in the intracellular region of AT1R allosterically potentiates AngII binding. LVV-H7 targets residues on intracellular loops 2 and 3 of AT1R, which are known binding sites of allosteric modulators in other GPCRs. Our data demonstrate the allosteric effect of LVV-H7 on AngII binding, which is consistent with the positive modulation of AT1R activity and signaling previously reported. This further supports the pharmacological targeting of AT1R by hemorphins, with implications in vascular and renal physiology.

Distinct but overlapping binding sites of agonist and antagonist at the relaxin family peptide 3 (RXFP3) receptor.

The relaxin-3 neuropeptide activates the relaxin family peptide 3 (RXFP3) receptor to modulate stress, appetite, and cognition. RXFP3 shows promise as a target for treating neurological disorders, but realization of its clinical potential requires development of smaller RXFP3-specific drugs that can penetrate the blood-brain barrier. Designing such drugs is challenging and requires structural knowledge of agonist- and antagonist-binding modes. Here, we used structure-activity data for relaxin-3 and a peptide RXFP3 antagonist termed R3 B1-22R to guide receptor mutagenesis and develop models of their binding modes. RXFP3 residues were alanine-substituted individually and in combination and tested in cell-based binding and functional assays to refine models of agonist and antagonist binding to active- and inactive-state homology models of RXFP3, respectively. These models suggested that both agonists and antagonists interact with RXFP3 via similar residues in their B-chain central helix. The models further suggested that the B-chain Trp27 inserts into the binding pocket of RXFP3 and interacts with Trp138 and Lys271, the latter through a salt bridge with the C-terminal carboxyl group of Trp27 in relaxin-3. R3 B1-22R, which does not contain Trp27, used a non-native Arg23 residue to form cation-π and salt-bridge interactions with Trp138 and Glu141 in RXFP3, explaining a key contribution of Arg23 to affinity. Overall, relaxin-3 and R3 B1-22R appear to share similar binding residues but may differ in binding modes, leading to active and inactive RXFP3 conformational states, respectively. These mechanistic insights may assist structure-based drug design of smaller relaxin-3 mimetics to manage neurological disorders.

Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3.

The neuropeptide relaxin-3 and its receptor relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake, and reward seeking. A linear relaxin-3 antagonist (R3 B1-22R) based on a modified and truncated relaxin-3 B-chain was recently developed. R3 B1-22R is unstructured in solution; thus, the binding conformation and determinants of receptor binding are unclear. Here, we have designed, chemically synthesized, and pharmacologically characterized more than 60 analogues of R3 B1-22R to develop an extensive understanding of its structure-activity relationships. We show that the key driver for affinity is the nonnative C-terminal Arg23 Additional contributors to binding include amino acid residues that are important also for relaxin-3 binding, including Arg12, Ile15, and Ile19 Intriguingly, amino acid residues that are not exposed in native relaxin-3, including Phe14 and Ala17, also interact with RXFP3. We show that R3 B1-22R has a propensity to form a helical structure, and modifications that support a helical conformation are functionally well-tolerated, whereas helix breakers such as proline residues disrupt binding. These data suggest that the peptide adopts a helical conformation, like relaxin-3, upon binding to RXFP3, but that its smaller size allows it to penetrate deeper into the orthosteric binding site, creating more extensive contacts with the receptor.

Random coil shifts of posttranslationally modified amino acids.

Most eukaryotic proteins are modified during and/or after translation, regulating their structure, function and localisation. The role of posttranslational modifications (PTMs) in both normal cellular processes and in diseases is already well recognised and methods for detection of PTMs and generation of specifically modified proteins have developed rapidly over the last decade. However, structural consequences of PTMs and their specific effects on protein dynamics and function are not well understood. Furthermore, while random coil NMR chemical shifts of the 20 standard amino acids are available and widely used for residue assignment, dihedral angle predictions and identification of structural elements or propensity, they are not available for most posttranslationally modified amino acids. Here, we synthesised a set of random coil peptides containing common naturally occurring PTMs and determined their random coil NMR chemical shifts under standardised conditions. We highlight unique NMR signatures of posttranslationally modified residues and their effects on neighbouring residues. This comprehensive dataset complements established random coil shift datasets of the 20 standard amino acids and will facilitate identification and assignment of posttranslationally modified residues. The random coil shifts will also aid in determination of secondary structure elements and prediction of structural parameters of proteins and peptides containing PTMs.

An Orbitide from Ratibida columnifera Seed Containing 16 Amino Acid Residues.

Cyclic peptides are abundant in plants and have attracted interest due to their bioactivity and potential as drug scaffolds. Orbitides are head-to-tail cyclic peptides that are ribosomally synthesized, post-translationally modified, and lack disulfide bonds. All known orbitides contain 5-12 amino acid residues. Here we describe PLP-53, a novel orbitide from the seed of Ratibida columnifera. PLP-53 consists of 16 amino acids, four residues larger than any known orbitide. NMR structural studies showed that, compared to previously characterized orbitides, PLP-53 is more flexible and, under the studied conditions, did not adopt a single ordered conformation based on analysis of NOEs and chemical shifts.

Two proteins for the price of one: Structural studies of the dual-destiny protein preproalbumin with sunflower trypsin inhibitor-1.

Seed storage proteins are both an important source of nutrition for humans and essential for seedling establishment. Interestingly, unusual napin-type 2S seed storage albumin precursors in sunflowers contain a sequence that is released as a macrocyclic peptide during post-translational processing. The mechanism by which such peptides emerge from linear precursor proteins has received increased attention; however, the structural characterization of intact precursor proteins has been limited. Here, we report the 3D NMR structure of the Helianthus annuus PawS1 (preproalbumin with sunflower trypsin inhibitor-1) and provide new insights into the processing of this remarkable dual-destiny protein. In seeds, PawS1 is matured by asparaginyl endopeptidases (AEPs) into the cyclic peptide SFTI-1 (sunflower trypsin inhibitor-1) and a heterodimeric 2S albumin. The structure of PawS1 revealed that SFTI-1 and the albumin are independently folded into well-defined domains separated by a flexible linker. PawS1 was cleaved in vitro with recombinant sunflower HaAEP1 and in situ using a sunflower seed extract in a way that resembled the expected in vivo cleavages. Recombinant HaAEP1 cleaved PawS1 at multiple positions, and in situ, its flexible linker was removed, yielding fully mature heterodimeric albumin. Liberation and cyclization of SFTI-1, however, was inefficient, suggesting that specific seed conditions or components may be required for in vivo biosynthesis of SFTI-1. In summary, this study has revealed the 3D structure of a macrocyclic precursor protein and provided important mechanistic insights into the maturation of sunflower proalbumins into an albumin and a macrocyclic peptide.

Alanine and Lysine Scans of the LL-37-Derived Peptide Fragment KR-12 Reveal Key Residues for Antimicrobial Activity.

The human host defence peptide LL-37 is a broad-spectrum antibiotic with immunomodulatory functions. Residues 18-29 in LL-37 have previously been identified as a minimal peptide (KR-12) that retains antibacterial activity with decreased cytotoxicity. In this study, analogues of KR-12 were generated by Ala and Lys scans to identify key elements for activity. These were tested against a panel of human pathogens and for membrane permeabilisation on liposomes. Replacements of hydrophobic and cationic residues with Ala were detrimental for antibiotic potency. Substitutions by Lys increased activity, as long as the increase in cationic density did not disrupt the amphiphilic disposition of the helical structure. Importantly, substitutions showed differential effects against different organisms. Replacement of Gln5 with Lys and Asp9 with Ala or Lys improved the broad-spectrum activity most, each resulting in up to an eightfold increase in potency against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The improved analogues displayed no significant toxicity against human cells, and thus, KR-12 is a tuneable template for antibiotic development.

Evolutionary origins of a bioactive peptide buried within Preproalbumin.

The de novo evolution of proteins is now considered a frequented route for biological innovation, but the genetic and biochemical processes that lead to each newly created protein are often poorly documented. The common sunflower (Helianthus annuus) contains the unusual gene PawS1 (Preproalbumin with SFTI-1) that encodes a precursor for seed storage albumin; however, in a region usually discarded during albumin maturation, its sequence is matured into SFTI-1, a protease-inhibiting cyclic peptide with a motif homologous to unrelated inhibitors from legumes, cereals, and frogs. To understand how PawS1 acquired this additional peptide with novel biochemical functionality, we cloned PawS1 genes and showed that this dual destiny is over 18 million years old. This new family of mostly backbone-cyclic peptides is structurally diverse, but the protease-inhibitory motif was restricted to peptides from sunflower and close relatives from its subtribe. We describe a widely distributed, potential evolutionary intermediate PawS-Like1 (PawL1), which is matured into storage albumin, but makes no stable peptide despite possessing residues essential for processing and cyclization from within PawS1. Using sequences we cloned, we retrodict the likely stepwise creation of PawS1's additional destiny within a simple albumin precursor. We propose that relaxed selection enabled SFTI-1 to evolve its inhibitor function by converging upon a successful sequence and structure.

Natural structural diversity within a conserved cyclic peptide scaffold.

We recently isolated and described the evolutionary origin of a diverse class of small single-disulfide bonded peptides derived from Preproalbumin with SFTI-1 (PawS1) proteins in the seeds of flowering plants (Asteraceae). The founding member of the PawS derived peptide (PDP) family is the potent trypsin inhibitor SFTI-1 (sunflower trypsin inhibitor-1) from Helianthus annuus, the common sunflower. Here we provide additional structures and describe the structural diversity of this new class of small peptides, derived from solution NMR studies, in detail. We show that although most have a similar backbone framework with a single disulfide bond and in many cases a head-to-tail cyclized backbone, they all have their own characteristics in terms of projections of side-chains, flexibility and physiochemical properties, attributed to the variety of their sequences. Small cyclic and constrained peptides are popular as drug scaffolds in the pharmaceutical industry and our data highlight how amino acid side-chains can fine-tune conformations in these promising peptides.