Α-helical Secondary Structure Research Articles

Bio-physical and computational studies on serum albumin / target protein binding of a potential anti-cancer agent

The successful evolution of an effective drug depends on its pharmacokinetics, efficiency and safety and these in turn depend on the drug-target/drug-carrier protein binding. This work, deals with the interaction of a pyridine derivative, 2-hydroxy-5-(4-methoxyphenyl)-6-phenylpyridine 3-carbonitrile (HDN) with serum albumins at physiological conditions utilizing the steady state and time-resolved fluorescence techniques by probing the emission behavior of Trp in BSA and HSA. In-silico studies revealed a combined static and dynamic quenching mechanism for the interactions. The binding studies suggests a spontaneous binding between HDN and the albumins with a moderate binding affinity (Kb ∼ 10–5 M-1) with a single class of binding site. The FRET mediated emission from HDN indicates preferential binding of HDN in subdomain IIA of the albumins with Trp residue in close proximity. Circular dichroism results indicate HDN induced conformational changes for BSA and HSA, but the α-helical secondary structure was well preserved even up to a concentration of 10 µM HDN. Moderate binding affinity of HDN with BSA and HSA and the unaltered secondary structure of proteins on binding propose the potential application of HDN as an efficient drug. The application of docking method on the affinity of HDN towards the proposed target/receptor is discussed.

Peptide stapling by late-stage Suzuki-Miyaura cross-coupling.

The development of peptide stapling techniques to stabilise α-helical secondary structure motifs of peptides led to the design of modulators of protein–protein interactions, which had been considered undruggable for a long time. We disclose a novel approach towards peptide stapling utilising macrocyclisation by late-stage Suzuki–Miyaura cross-coupling of bromotryptophan-containing peptides of the catenin-binding domain of axin. Optimisation of the linker length in order to find a compromise between both sufficient linker rigidity and flexibility resulted in a peptide with an increased α-helicity and enhanced binding affinity to its native binding partner β-catenin. An increased proteolytic stability against proteinase K has been demonstrated.

Peptide-Based Dual HIV and Coronavirus Entry Inhibitors.

The continued HIV/AIDS epidemic worldwide and the battle against emerging infectious diseases caused by coronaviruses underscore the need for the development of an ever-expanding repertoire of antiviral drugs. Entry inhibitors are of particular interest because of their potential to be used as therapeutic or prophylactic treatments for blocking viral invasion. HIV and coronaviruses utilize class I fusion proteins to facilitate their entry and membrane fusion. Discovery of a common hexameric coiled-coil fusion complex resulting from the packing of three C-terminal heptad repeat region from the fusion-mediating subunit of viral fusion proteins against trimeric coiled-coil made up by their N-terminal heptad repeat prompted the search for peptides mimicking the heptad repeat regions that could potentially inhibit viral entry. This has led to the development of effective peptides that are specific to the virus that is developed for. In this review, we focus on peptide-based entry dual inhibitors that block fusion process not only of HIV but also coronaviruses through interrupting their fusogenic six-helical bundle core and which hopefully will help to gain insight into the α-helical secondary structure- and coiled-coil superstructure-based strategies to design entry inhibitors with broad-spectrum antiviral activity against enveloped viruses with class I fusion proteins.

Effect of Divalent Metal Ion on the Structure, Stability and Function of Klebsiella pneumoniae Nicotinate-Nucleotide Adenylyltransferase: Empirical and Computational Studies.

The continuous threat of drug-resistant Klebsiella pneumoniae justifies identifying novel targets and developing effective antibacterial agents. A potential target is nicotinate nucleotide adenylyltransferase (NNAT), an indispensable enzyme in the biosynthesis of the cell-dependent metabolite, NAD+. NNAT catalyses the adenylation of nicotinamide/nicotinate mononucleotide (NMN/NaMN), using ATP to form nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD). In addition, it employs divalent cations for co-substrate binding and catalysis and has a preference for different divalent cations. Here, the biophysical structure of NNAT from K. pneumoniae (KpNNAT) and the impact of divalent cations on its activity, conformational stability and substrate-binding are described using experimental and computational approaches. The experimental study was executed using an enzyme-coupled assay, far-UV circular dichroism, extrinsic fluorescence spectroscopy, and thermal shift assays, alongside homology modelling, molecular docking, and molecular dynamic simulation. The structure of KpNNAT revealed a predominately α-helical secondary structure content and a binding site that is partially hydrophobic. Its substrates ATP and NMN share the same binding pocket with similar affinity and exhibit an energetically favourable binding. KpNNAT showed maximum activity and minimal conformational changes with Mg2+ as a cofactor compared to Zn2+, Cu2+ and Ni2+. Overall, ATP binding affects KpNNAT dynamics, and the dynamics of ATP binding depend on the presence and type of divalent cation. The data obtained from this study would serve as a basis for further evaluation towards designing structure-based inhibitors with therapeutic potential.

Spectroscopic evidence of tetanus toxin translocation domain bilayer-induced refolding and insertion

Tetanus neurotoxin (TeNT) is an A-B toxin with three functional domains: endopeptidase, translocation (HCT), and receptor binding. Endosomal acidification triggers HCT to interact with and insert into the membrane, translocating the endopeptidase across the bilayer. Although the function of HCT is well defined, the mechanism by which it accomplishes this task is unknown. To gain insight into the HCT membrane interaction on both local and global scales, we utilized an isolated, beltless HCT variant (bHCT), which retained the ability to release potassium ions from vesicles. To examine which bHCT residues interact with the membrane, we widely sampled the surface of bHCT using 47 single-cysteine variants labeled with the environmentally sensitive fluorophore NBD. At neutral pH, no interaction was observed for any variant. In contrast, all NBD-labeled positions reported environmental change in the presence of acidic pH and membranes containing anionic lipids. We then examined the conformation of inserted bHCT using circular dichroism and intrinsic fluorescence. Upon entering the membrane, bHCT retained predominantly α-helical secondary structure, whereas the tertiary structure exhibited substantial refolding. The use of lipid-attached quenchers revealed that at least one of the three tryptophan residues penetrated deep into the hydrocarbon core of the membrane, suggesting formation of a bHCT transmembrane conformation. The possible conformational topology was further explored with the hydropathy analysis webtool MPEx, which identified a large, potential α-helical transmembrane region. Altogether, the spectroscopic evidence supports a model in which, upon acidification, the majority of TeNT bHCT entered the membrane with a concurrent change in tertiary structure.

Citrullination Alters the Antibacterial and Anti-Inflammatory Functions of the Host Defense Peptide Canine Cathelicidin K9CATH In Vitro.

K9CATH is the sole cathelicidin in canines (dogs) and exhibits broad antimicrobial activity against both Gram-positive and Gram-negative bacteria. K9CATH also modulates inflammatory responses and binds to LPS. These activities depend on the secondary structure and a net-positive charge of the peptide. Peptidylarginine deiminases (PAD) convert cationic peptidyl arginine to neutral citrulline. Thus, we hypothesized that citrullination is a biologically relevant modification of the peptide that would reduce the antibacterial and LPS-binding activities of K9CATH. Recombinant PAD2 and PAD4 citrullinated K9CATH to various extents and circular dichroism spectroscopy revealed that both native and citrullinated K9CATH exhibited similar α-helical secondary structures. Notably, citrullination of K9CATH reduced its bactericidal activity, abolished its ability to permeabilize the membrane of Gram-negative bacteria and reduced the hemolytic capacity. Electron microscopy showed that citrullinated K9CATH did not cause any morphological changes of Gram-negative bacteria, whereas the native peptide caused clear alterations of membrane integrity, concordant with a rapid bactericidal effect. Finally, citrullination of K9CATH impaired its capacity to inhibit LPS-mediated release of proinflammatory molecules from mouse and canine macrophages. In conclusion, citrullination attenuates the antibacterial and the LPS-binding properties of K9CATH, demonstrating the importance of a net positive charge for antibacterial lysis of bacteria and LPS-binding effects and suggests that citrullination is a means to regulate cathelicidin activities.

Ubiquitin forms conventional decorated micelle structures with sodium dodecyl sulfate at saturation

The anionic surfactant sodium dodecyl sulfate (SDS) interacts strongly with most globular proteins and denatures and unfolds them. While scattering studies using X-rays and neutrons have shown that this denaturation generally leads to protein-decorated SDS micelles, a different SDS-decorated polypeptide model has recently been suggested for complexes between SDS and Ubiquitin (UBI), in which individual SDS molecules are distributed on a partially stretched protein. To resolve this apparent discrepancy, we have investigated the SDS-UBI system by a number of complementary techniques. Small-angle X-ray scattering (SAXS) provides the overall structure of the SDS-UBI complexes, Tyr fluorescence and circular dichroism follow changes in tertiary and secondary structure, and isothermal titration calorimetry determines the stoichiometries of complexes and the amount of free SDS as a function of [SDS]. At low [SDS], UBI preserves its folded structure but dimerizes to a small extent. At 4 SDS per UBI, a complex is formed with two UBI and a small shared SDS cluster with 8 SDS molecules. In these complexes UBI preserves most of its native fold. At 10–12 SDS per UBI, which remains below the critical micelle concentration under our conditions, UBI-covered SDS micelles form with four UBIs around a core of 40 SDSs. This implies a protein-assisted micellization and an associated unfolding of UBI involving a change from mainly β-strands to mainly α-helical secondary structure. As [SDS] is increased, the complex gradually changes so that finally only one UBI covers one micelle with a similar number of SDS molecules at SDS saturation. Thus, we conclude that SDS unfolds UBI by mechanisms very similar to those observed for other globular proteins, leading to a protein-decorated SDS micelle rather than an SDS-decorated unfolded polypeptide chain.

The leucine zipper EF-hand containing transmembrane protein-1 EF-hand is a tripartite calcium, temperature, and pH sensor.

Leucine Zipper EF-hand containing transmembrane protein-1 (LETM1) is an inner mitochondrial membrane protein that mediates mitochondrial calcium (Ca2+ )/proton exchange. The matrix residing carboxyl (C)-terminal domain contains a sequence identifiable EF-hand motif (EF1) that is highly conserved among orthologues. Deletion of EF1 abrogates LETM1 mediated mitochondrial Ca2+ flux, highlighting the requirement of EF1 for LETM1 function. To understand the mechanistic role of this EF-hand in LETM1 function, we characterized the biophysical properties of EF1 in isolation. Our data show that EF1 exhibits α-helical secondary structure that is augmented in the presence of Ca2+ . Unexpectedly, EF1 features a weak (~mM), but specific, apparent Ca2+ -binding affinity, consistent with the canonical Ca2+ coordination geometry, suggested by our solution NMR. The low affinity is, at least in part, due to an Asp at position 12 of the binding loop, where mutation to Glu increases the affinity by ~4-fold. Further, the binding affinity is sensitive to pH changes within the physiological range experienced by mitochondria. Remarkably, EF1 unfolds at high and low temperatures. Despite these unique EF-hand properties, Ca2+ binding increases the exposure of hydrophobic regions, typical of EF-hands; however, this Ca2+ -induced conformational change shifts EF1 from a monomer to higher order oligomers. Finally, we showed that a second, putative EF-hand within LETM1 is unreactive to Ca2+ either in isolation or tandem with EF1. Collectively, our data reveal that EF1 is structurally and biophysically responsive to pH, Ca2+ and temperature, suggesting a role as a multipartite environmental sensor within LETM1.

Tomato calmodulin-like protein SlCML37 is a calcium (Ca2+) sensor that interacts with proteasome maturation factor SlUMP1 and plays a role in tomato fruit chilling stress tolerance

Calmodulin-like proteins (CMLs), as well as their targets, play significant roles in various key developmental and stress responses in the plant. In tomato (Solanum lycopersicum), there are at least 52 CML genes in its genome. However, most of their functions are not well known, especially in response to cold stress. Here, we investigated SlCML37 biochemical and structural characteristics, including a typical α-helical secondary structure and exposing its hydrophobic regions after binding to Ca2+. Then we certificated that SlCML37 protein could physically interact with SlUMP1 by using yeast two-hybrid, bimolecular florescence complementation (BiFC) and GST pull-down assays. Further analysis showed that SlCML37-transgenic tomato fruit conferred significantly improved tolerance to chilling stress. This study indicates a possible role of calmodulin-like protein-mediated proteasome assemble in the regulation of plant cold response.

Tyrosine phosphorylation regulates hnRNPA2 granule protein partitioning and reduces neurodegeneration.

mRNA transport in neurons requires formation of transport granules containing many protein components, and subsequent alterations in phosphorylation status can release transcripts for translation. Further, mutations in a structurally disordered domain of the transport granule protein hnRNPA2 increase its aggregation and cause hereditary proteinopathy of neurons, myocytes, and bone. We examine in vitro hnRNPA2 granule component phase separation, partitioning specificity, assembly/disassembly, and the link to neurodegeneration. Transport granule components hnRNPF and ch‐TOG interact weakly with hnRNPA2 yet partition specifically into liquid phase droplets with the low complexity domain (LC) of hnRNPA2, but not FUS LC. In vitro hnRNPA2 tyrosine phosphorylation reduces hnRNPA2 phase separation, prevents partitioning of hnRNPF and ch‐TOG into hnRNPA2 LC droplets, and decreases aggregation of hnRNPA2 disease variants. The expression of chimeric hnRNPA2 D290V in Caenorhabditis elegans results in stress‐induced glutamatergic neurodegeneration; this neurodegeneration is rescued by loss of tdp‐1, suggesting gain‐of‐function toxicity. The expression of Fyn, a tyrosine kinase that phosphorylates hnRNPA2, reduces neurodegeneration associated with chimeric hnRNPA2 D290V. These data suggest a model where phosphorylation alters LC interaction specificity, aggregation, and toxicity.

Synthesis and Structural Stability of α-Helical Gold(I)-Metallopeptidesy

AbstractThe synthesis of hexa- and dodecapeptides functionalized with two Au(I)–phosphine complexes is reported. The high stability of the Au(I)–phosphine bond allowed orthogonal peptide-protecting-group chemistry, even when using hard Lewis acids like boron tribromide. This enabled the preparation of an Fmoc-protected lysine derivative carrying the Au(I) complex in a side chain, which was used in standard Fmoc-based solid-phase peptide synthesis protocols. Alanine and leucine repeats in the metallododecapeptide formed α-helical secondary structures in 2,2,2-trifluoroethanol–H2O and 1,1,1,3,3,3-hexafluoroisopropanol–H2O mixtures with high thermal stability, as shown by temperature-dependent CD spectroscopy studies.

Human Serum Albumin Binds Native Insulin and Aggregable Insulin Fragments and Inhibits Their Aggregation.

The purpose of this study was to investigate whether Human Serum Albumin (HSA) can bind native human insulin and its A13–A19 and B12–B17 fragments, which are responsible for the aggregation of the whole hormone. To label the hormone and both hot spots, so that their binding positions within the HSA could be identified, 4-(1-pyrenyl)butyric acid was used as a fluorophore. Triazine coupling reagent was used to attach the 4-(1-pyrenyl)butyric acid to the N-terminus of the peptides. When attached to the peptides, the fluorophore showed extended fluorescence lifetimes in the excited state in the presence of HSA, compared to the samples in buffer solution. We also analyzed the interactions of unlabeled native insulin and its hot spots with HSA, using circular dichroism (CD), the microscale thermophoresis technique (MST), and three independent methods recommended for aggregating peptides. The CD spectra indicated increased amounts of the α-helical secondary structure in all analyzed samples after incubation. Moreover, for each of the two unlabeled hot spots, it was possible to determine the dissociation constant in the presence of HSA, as 14.4 µM (A13–A19) and 246 nM (B12–B17). Congo Red, Thioflavin T, and microscopy assays revealed significant differences between typical amyloids formed by the native hormone or its hot-spots and the secondary structures formed by the complexes of HSA with insulin and A13–A19 and B12–B17 fragments. All results show that the tested peptide-probe conjugates and their unlabeled analogues interact with HSA, which inhibits their aggregation.

Synergistic Biophysical Techniques Reveal Structural Mechanisms of Engineered Cationic Antimicrobial Peptides in Lipid Model Membranes.

In the quest for new antibiotics, two novel engineered cationic antimicrobial peptides (eCAPs) have been rationally designed. WLBU2 and D8 (all 8 valines are the d-enantiomer) efficiently kill both Gram-negative and -positive bacteria, but WLBU2 is toxic and D8 nontoxic to eukaryotic cells. We explore protein secondary structure, location of peptides in six lipid model membranes, changes in membrane structure and pore evidence. We suggest that protein secondary structure is not a critical determinant of bactericidal activity, but that membrane thinning and dual location of WLBU2 and D8 in the membrane headgroup and hydrocarbon region may be important. While neither peptide thins the Gram-negative lipopolysaccharide outer membrane model, both locate deep into its hydrocarbon region where they are primed for self-promoted uptake into the periplasm. The partially α-helical secondary structure of WLBU2 in a red blood cell (RBC) membrane model containing 50 % cholesterol, could play a role in destabilizing this RBC membrane model causing pore formation that is not observed with the D8 random coil, which correlates with RBC hemolysis caused by WLBU2 but not by D8.

Accelerated Protein Folding Using Greedy-Proximal A.

The protein folding problem has been studied in the field of molecular biophysics and biochemistry for many years. Even small changes in folding patterns may lead to serious diseases such as Alzheimer's or Parkinson's where proteins are folded either too quickly or too slowly. Molecular dynamics (MD) is one of the tools used to understand how proteins fold into native conformations. While it captures sequences of conformations that lead over time to the folded state, limitations in simulation timescales remain problematic. Although many approaches have been suggested to speed up the simulation process using rapid changes in temperature or pressure, we propose a rational approach, Greedy-proximal A* (GPA*), derived from path finding algorithms to explore the supposed shortest path folding pathway from the unfolded to a given folded conformation. We introduce several new protein structure comparison metrics based on the contact map distance to help mitigate the challenges faced by "standard" metrics. We test our approach on proteins which represent the two main types of secondary structure: (a) the Trp-cage miniprotein construct TC5b (1L2Y) which is a short, fast-folding protein that represents an α-helical secondary structure formed because of a locked tryptophan in the middle, (b) the immunoglobulin binding domain of the streptococcal protein G (1GB1), containing an α-helix and several β-sheets, and (c) the chicken villin subdomain HP-35, N68H protein (1YRF)-one of the fastest folding proteins which forms three α-helices. We compare our algorithm to replica-exchange MD and steered MD methods which represent the main algorithms used for accelerating folding proteins with MD. We find that GPA* not only reduces the computational time needed to obtain the folded conformation without adding artificial energy bias but also makes it possible to generate trajectories which contain minimal motions needed for the folding transition.

Nitric Oxide-Biosignal-Responsive Polypeptide Nanofilaments.

Nitric oxide (NO) is one of the three gasotransmitters in living organisms that relates to numerous biological signaling processes. The quest to smart molecular self-assembled systems with ideal NO-responsivity enables precise therapy and diagnosis of NO-associated diseases. Here we report a class of amphiphilic polypeptide containing an o-phenylenediamine-functionalized poly(l-glutamate) segment (PEOPA) that can respond to NO biosignal with extraordinary sensitivity and selectivity. Such polypeptides can further form a rigid filament nanostructure via the synergism of an α-helical secondary structure of PEOPA blocks and H-bonding between interchain OPA side groups. A biologically relevant level of NO can cleave the filament-forming OPA motifs, thus, leading to a dissociation of the assemblies. This NO-responsive disassembly behavior makes these polypeptide nanofilaments as NO-activated nanocarriers, which hold promise for NO-pathological milieu-specific drug delivery.

Uncovering Order within the Disorder: Redefining IA3's Intrinsically Disordered Properties

IA3 is a 68 residue intrinsically disordered protein (IDP), found in Saccharomyces cerevisiae, that adopts N-terminal α-helical secondary structure when bound to yeast proteinase A (YPRA). The α-helical transition of IA3 can be stabilized in the absence of YPRA, by using 2,2,2-trifluoroethanol (TFE). Circular dichroism spectroscopy (CD) was used to characterize the unstructured-to-structured transition of wild type IA3 along with residue substituted mutants of this protein. These mutational studies showed TFE induced impacts on helical intensity in the structured state, but also uncovered variations in the unstructured, or disordered state of the protein. The observed structural differences in the presence of TFE indicated changes to helical intensity, however, we also found differences in residual order in the unfolded states, in the absence of TFE. Residual order of an IDP in an unfolded state suggests a distinction between a disordered and denatured protein, where residual order can be removed, an idea of “order within the disorder”. We found that the addition of differing amounts of urea removes the residual structure where wild type and all variants converge to a similar degree of un-folding, indicating a thermodynamic difference in the order of these unstructured ensembles. Dynamic light scattering (DLS) along with differential scanning calorimetry (DSC) results will be presented to further analyze changes in the disordered structure through thermal melting, and urea-induced denaturation. DLS and DSC results will characterize the changes in hydrodynamic radius, along with a determined melting temperature, the enthalpy of unfolding, and heat capacity change upon denaturation of the disordered state. These combined techniques will probe the presence of an energy barrier in the disordered state of IA3 that could indicate an ability to disrupt the “order within the disorder”.

Disorder for Dummies: Functional Mutagenesis of Transient Helical Segments in Disordered Proteins.

Most cytosolic eukaryotic proteins contain a mixture of ordered and disordered regions. Disordered regions facilitate cell signaling by concentrating sites for posttranslational modifications and protein-protein interactions into arrays of short linear motifs that can be reorganized by RNA splicing. The evolution of disordered regions looks different from their ordered counterparts. In some cases, selection is focused on maintaining protein binding interfaces and PTM sites, but sequence heterogeneity is common. In other cases, simple properties like charge, length, or end-to-end distance are maintained. Many disordered protein binding sites contain some transient secondary structure that may resemble the structure of the bound state. α-Helical secondary structure is common and a wide range of fractional helicity is observed in different disordered regions. Here we provide a simple protocol to identify transient helical segments and design mutants that can change their structure and function.

Engineering Helical Modular Polypeptide-Based Hydrogels as Synthetic Extracellular Matrices for Cell Culture.

Expanding the toolkit of modular and functional synthetic material systems for biomimetic extracellular matrices (ECMs) is needed for achieving more predictable and characterizable cell culture. In the present study, we engineered a synthetic hydrogel system incorporating poly(γ-propargyl-l-glutamate) (PPLG), an N-carboxy anhydride polypeptide with a unique α-helical secondary structure. PPLG macromers were cross-linked into poly(ethylene glycol) (PEG) networks to form hybrid polypeptide-PEG hydrogels. We compared the properties of PPLG-PEG to systems where the PPLG macromers were replaced with 8-arm PEG or poly(γ-propargyl-d,l-glutamate) (PPDLG), which has a flexible random-coil conformation. We evaluated each hydrogel system as synthetic ECMs for two-dimensional (2D) endothelial cell culture. Cells on PPLG-PEG displayed superior attachment and spreading at comparable adhesion ligand incorporation concentrations, demonstrating the unique benefit of combining the more rigid and hydrophobic α-helical PPLG within the more flexible and hydrophilic PEG matrix. The modular PPLG macromer is a promising building block for developing other types of PPLG-based hydrogels with favorable and tunable properties.

Structure-Activity Study of an All-d Antimicrobial Octapeptide D2D.

The increasing emergence of multi-drug resistant bacteria is a serious threat to public health worldwide. Antimicrobial peptides have attracted attention as potential antibiotics since they are present in all multicellular organisms and act as a first line of defence against invading pathogens. We have previously identified a small all-d antimicrobial octapeptide amide kk(1-nal)fk(1-nal)k(nle)-NH2 (D2D) with promising antimicrobial activity. In this work, we have performed a structure-activity relationship study of D2D based on 36 analogues aimed at discovering which elements are important for antimicrobial activity and toxicity. These modifications include an alanine scan, probing variation of hydrophobicity at lys5 and lys7, manipulation of amphipathicity, N-and C-termini deletions and lys-arg substitutions. We found that the hydrophobic residues in position 3 (1-nal), 4 (phe), 6 (1-nal) and 8 (nle) are important for antimicrobial activity and to a lesser extent cationic lysine residues in position 1, 2, 5 and 7. Our best analogue 5, showed MICs of 4 µg/mL against A. baumannii, E. coli, P. aeruginosa and S. aureus with a hemolytic activity of 47% against red blood cells. Furthermore, compound 5 kills bacteria in a concentration-dependent manner as shown by time-kill kinetics. Circular dichroism (CD) spectra of D2D and compounds 1–8 showed that they likely fold into α-helical secondary structure. Small angle x-ray scattering (SAXS) experiments showed that a random unstructured polymer-like chains model could explain D2D and compounds 1, 3, 4, 6 and 8. Solution structure of compound 5 can be described with a nanotube structure model, compound 7 can be described with a filament-like structure model, while compound 2 can be described with both models. Lipid interaction probed by small angle X-ray scattering (SAXS) showed that a higher amount of compound 5 (~50–60%) inserts into the bilayer compared to D2D (~30–50%). D2D still remains the lead compound, however compound 5 is an interesting antimicrobial peptide for further investigations due to its nanotube structure and minor improvement to antimicrobial activity compared to D2D.

Ribbon α-Conotoxin KTM Exhibits Potent Inhibition of Nicotinic Acetylcholine Receptors.

KTM is a 16 amino acid peptide with the sequence WCCSYPGCYWSSSKWC. Here, we present the nuclear magnetic resonance (NMR) structure and bioactivity of this rationally designed α-conotoxin (α-CTx) that demonstrates potent inhibition of rat α3β2-nicotinic acetylcholine receptors (rα3β2-nAChRs). Two bioassays were used to test the efficacy of KTM. First, a qualitative PC12 cell-based assay confirmed that KTM acts as a nAChR antagonist. Second, bioactivity evaluation by two-electrode voltage clamp electrophysiology was used to measure the inhibition of rα3β2-nAChRs by KTM (IC50 = 0.19 ± 0.02 nM), and inhibition of the same nAChR isoform by α-CTx MII (IC50 = 0.35 ± 0.8 nM). The three-dimensional structure of KTM was determined by NMR spectroscopy, and the final set of 20 structures derived from 32 distance restraints, four dihedral angle constraints, and two disulfide bond constraints overlapped with a mean global backbone root-mean-square deviation (RMSD) of 1.7 ± 0.5 Å. The structure of KTM did not adopt the disulfide fold of α-CTx MII for which it was designed, but instead adopted a flexible ribbon backbone and disulfide connectivity of C2–C16 and C3–C8 with an estimated 12.5% α-helical content. In contrast, α-CTx MII, which has a native fold of C2–C8 and C3–C16, has an estimated 38.1% α-helical secondary structure. KTM is the first reported instance of a Framework I (CC-C-C) α-CTx with ribbon connectivity to display sub-nanomolar inhibitory potency of rα3β2-nAChR subtypes.