Ile Residues Research Articles

Nuclear Magnetic Resonance at the Interface: Identifying Preferred Binding Regions in Multimodal Cation Exchange Chromatography using Functionalized Nanoparticles

The Fc domain is an important component of complex molecules such as monoclonal antibodies (mAb) based biologics as well as fusion proteins and is highly conserved across a given class of mAbs. Recent work in our lab has shown the importance of specific domains towards binding for mAbs to multimodal (MM) cation exchange chromatographic systems. In this collaborative work, we employ a combination of Nuclear Magnetic Resonance (NMR) spectroscopy and Umbrella sampling simulations to develop a fundamental understanding of how multimodal ligands and surfaces interact with the Fc domain. We performed NMR titration experiments with isotopically labeled Fc and ∼15 nm diameter gold nanoparticles (Au NPs) functionalized with Self Assembled Monolayers (SAMs) presenting different MM ligands at different ligand densities. The results showed the interface of CH2 and CH3 domains and the hinge region to be preferred binding regions for interaction of Fc to MM ligand surfaces. These flexible regions on the protein surface which are rich in positive and aliphatic residues are important for the binding of Fc to MM ligand surfaces. Further, to shed light on the binding mechanisms, we performed umbrella sampling with the Fc held at key orientations above a SAM surface presenting MM ligands at relevant ligand densities similar to those tested experimentally. These simulations suggested that as the Fc was brought close to the surface, the residues near the hinge region first made contact with the surface followed by a tighter binding facilitated by interaction of His, Val, Leu and Ile residues at the interface of the CH2 and CH3 domains. This combined experimental and simulations approach enabled us to develop a molecular level understanding of the binding for complex biological products in MM CEX systems.

Analysis of interleukin 23 and 7G10 interactions for computational design of lead antibodies against immune-mediated inflammatory diseases

Wealth of structural data on theurapeutic targets in complex with monoclonal antibodies (mAbs) and advances in molecular modeling algorithms present exciting opportunities in the field of novel biologic design. Interleukin 23 (IL23), a well-known drug target for autoimmune diseases, in complex with mAb 7G10 offers prospect to design potent lead antibodies by traversing the complete epitope–paratope interface. Herein, key interactions aiding antibody-based neutralization in IL23–7G10 complex are resolute through PyMOL, LigPlot+, Antibody i-Patch, DiscoTope and FoldX. Six amino acids Ser31, Val33, Asn55, Lys59 in heavy chain and His34, Ser93 in light chain are subjected to in silico mutagenesis with residues Met, Trp, Ile, Leu and Arg. A set of 431 mutant macromolecules are outlined. Binding affinities of these molecules with IL23 are estimated through protein–protein docking by employing ZDOCK, ClusPro and RosettaDock. Subsequently, the macromolecules revealed comparable result with 7G10 are cross validated through binding free-energy calculations by applying Molecular Mechanics/Poisson Boltzman Surface Area method in CHARMM. Thirty nine designed theoretical antibodies showed improved outcome in all evaluations; from these, top 10 molecules showed at least nine unit better binding affinity compared to the known mAb. These molecules have the potential to act as lead antibodies. Subsequent molecular dynamics simulations too favored prospective of best ranked molecule to have therapeutic implications in autoimmune and inflammatory diseases.Abbreviations: IL23: interleukin 23; IL17: interleukin17; Ab: antibody; Ag: antigen; mAbs: monoclonal antibodies; STAT3: signal transducer and activator of transcription 3; STAT4: signal transducer and activator of transcription 4; PDB: protein databank; MM/PBSA: molecular mechanics Poisson-Boltzmann surface area; Ag–Ab: antigen– antibody complex; SPC/E: extended simple point charge; SD: steepest descents; PME: particle mesh ewald; dG: binding free energies; Fv: variable fragment

Engineering potent mesotrypsin inhibitors based on the plant-derived cyclic peptide, sunflower trypsin inhibitor-1

Plants produce a diverse range of peptides and proteins that inhibit the activity of different serine proteases. The value of these inhibitors not only stems from their native role(s) in planta, but they are also regarded as promising templates for inhibitor engineering. Interest in this field has grown rapidly in recent years, particularly for therapeutic applications. The serine protease mesotrypsin has been implicated in several cancers, but is a challenging target for inhibitor engineering as a number of serine protease inhibitors that typically display broad-range activity show limited activity against mesotrypsin. In this study, we use a cyclic peptide isolated from sunflower seeds, sunflower trypsin inhibitor-1 (SFTI-1), as a scaffold for engineering potent mesotrypsin inhibitors. SFTI-1 comprises 14-amino acids and is a potent inhibitor of human cationic trypsin (Ki = 30 ± 0.8 pM) but shows 165,000-fold weaker activity against mesotrypsin (Ki = 4.96 ± 0.2 μM). Using an inhibitor library based on SFTI-1, we show that the inhibitor's P2′ residue (Ile) is a key contributor to SFTI-1's limited activity against mesotrypsin. Substituting P2′ Ile with chemically diverse amino acids, including non-canonical aromatic residues, produced new inhibitor variants that maintained a similar structure to SFTI-1 and showed marked improvements in activity (exceeding 100-fold). An assessment of the activity of the new inhibitors against closely-related trypsin paralogs revealed that the improved activity against mesotrypsin was accompanied by a loss in activity against off-target proteases, such that several engineered variants showed comparable activity against mesotrypsin and human cationic trypsin. Together, these findings identify potent mesotrypsin inhibitors that are suitable for further optimisation studies and demonstrate the potential gains in activity and selectivity that can be achieved by optimising the P2′ residue, particularly for engineered SFTI-based inhibitors.

Relative role(s) of leucine versus isoleucine in the folding of membrane proteins

AbstractLarge, hydrophobic residues (isoleucine, leucine, and valine) dominate sequences of transmembrane (TM) helices in membrane proteins (total ∼34%), but their relative roles in mediating the biologically relevant protein–lipid and protein–protein interactions have not been systematically evaluated. Here we have synthesized Leu‐containing Lys‐tagged hydrophobic peptides of identical composition, where sequences have been designed with their Leu residues either scrambled (sequence KKKLAASALAAAWLAALALSAAKKK); clustered (KKKAAASAALLLWLLAAAASAAKKK); or “lipopathic” (all Leu on one helical face) (KKKAAASLAALLWALLAAASAAKKK). These peptides were compared by several biophysical/biochemical techniques to the corresponding set of peptides where the Leu residues are replaced by the isosteric Ile residues. Circular dichroism spectra showed that all peptides were helical in POPC liposomes, as confirmed by blue shifts in Trp fluorescence spectra, notably with the Ile‐lipopathic peptide displaying increased Trp burial versus its Leu counterpart. Quenching experiments with a dibromo‐PC lipid indicated deeper membrane penetration of the Ile versus the Leu lipopathic peptide—a result supported by protease degradation assays where Ile peptides reconstituted into lipid bilayers were significantly more protected from the protease than the Leu peptides. Assessment of Trp blue shifts in the presence of lipid bilayers of varied lipid packing indicated that Leu/Ile peptide interactions are dependent on lipid composition. The overall results suggest that two main interactions tend to dominate Leu and Ile interactions within the membrane: (1) hydrophobic interactions between amino acid side chains and the surrounding lipid; and (2) degree of disruption of lipid–lipid packing. This “battle of giants” likely underlies the specific role(s) that Leu and Ile will play in the folding of a given membrane protein.

Genome mining for peptidases in heat-tolerant and mesophilic fungi and putative adaptations for thermostability

BackgroundPeptidases (EC 3.4) consist of a large group of hydrolytic enzymes that catalyze the hydrolysis of proteins accounting for approximately 65% of the total worldwide enzyme production. Peptidases from thermophilic fungi have adaptations to high temperature that makes them adequate for biotechnological application. In the present study, we profiled the genomes of heat-tolerant fungi and phylogenetically related mesophilic species for genes encoding for peptidases and their putative adaptations for thermostability.ResultsWe generated an extensive catalogue of these enzymes ranging from 241 to 820 peptidase genes in the genomes of 23 fungi. Thermophilic species presented the smallest number of peptidases encoding genes in relation to mesophilic species, and the peptidases families with a greater number of genes were the most affected. We observed differences in peptidases in thermophilic species in comparison to mesophilic counterparts, at (i) the genome level: a great reduction in the number of peptidases encoding genes that harbored a higher number of copies; (ii) in the primary protein structure: shifts in proportion of single or groups of amino acids; and (iii) in the three-dimensional structure: reduction in the number of internal cavities. Similar results were reported for extremely thermophilic proteins, but here we show for the first time that several changes also occurred on the moderate thermophilic enzymes of fungi. In regards to the amino acids composition, peptidases from thermophilic species in relation to the mesophilic ones, contained a larger proportion of Ala, Glu, Gly, Pro, Arg and Val residues and a lower number of Cys, His, Ile, Lys, Met, Asn, Gln, Ser, Thr and Trp residues (P < 0.05). Moreover, we observed an increase in the proportion of hydrophobic and charged amino acids and a decrease in polar amino acids.ConclusionsAlthough thermophilic fungi present less genes encoding for peptidases, these have adaptations that could play a role in thermal resistance from genome to protein structure level.

Central β-turn increases the cell selectivity of imperfectly amphipathic α-helical peptides

Although membrane lytic antimicrobial peptides (AMPs) show enormous potential for addressing mounting global antibiotic resistance, therapeutic applications are hindered by their weak antimicrobial activity, high toxicity, salt sensitivity and poor understanding of structure-activity relationships. To investigate the effects of different parameters on the biological activities of AMPs, a rational approach was adopted to design a series of short cationic α-helical peptides comprising the Ac-WxKyWxzzyKxWyK-NH2 sequence, where x: cationic residues (Arg or Lys), y: hydrophobic residues (Ala, Val, Ile or Leu), and zz: β-turn (rigid D-Pro-Gly turn or flexible Gly-Gly turn). The peptides showed a more helical structure as the concentration of membrane-mimetic solution increased. The peptide RL with a central D-Pro-Gly turn (x: Arg, y: Lys, zz = D-Pro-Gly) exhibited broad-spectrum antimicrobial activities (2–8 μM) against ten types of clinically relevant microorganisms and even maintained its activity in the presence of physiological salts and showed excellent selectivity toward bacterial cells over human red blood cells and mammalian cells. However, the toxicity was increased after the removal of D-Pro-Gly turn. Additionally, the bactericidal activity was reduced when the D-Pro-Gly turn was replaced by a Gly-Gly turn. Fluorescence spectroscopy and electron microscopy analyses indicated that RL and its derivatives killed microbial cells by permeabilizing the cell membrane and damaging membrane integrity. In conclusion, these findings clearly generalized a potential method for designing or optimizing AMPs, and the peptide RL is a promising therapeutic candidate to combat antibiotic resistance. Statement of SignificanceWe proposed a rational approach to design imperfectly amphiphilic peptides and identified RL (Ac-WRKLWRpGLKRWLK-NH2) in particular that shows strong antibacterial properties, low toxicity and high salt resistance. The β-turn unit inserted into the central position of cationic α-helical peptides, especially the D-Pro-Gly turn, significantly increase the cell selectivity of the synthetic amphiphiles. The findings demonstrate a potential method for designing and/or optimizing AMPs, which would facilitate the development of strategies to design peptide-based antimicrobial biomaterials in a variety of biotechnological and clinical applications.

Structural analyses of adenylate kinases from Antarctic and tropical fishes for understanding cold adaptation of enzymes

Psychrophiles are extremophilic organisms capable of thriving in cold environments. Proteins from these cold-adapted organisms can remain physiologically functional at low temperatures, but are structurally unstable even at moderate temperatures. Here, we report the crystal structure of adenylate kinase (AK) from the Antarctic fish Notothenia coriiceps, and identify the structural basis of cold adaptation by comparison with homologues from tropical fishes including Danio rerio. The structure of N. coriiceps AK (AKNc) revealed suboptimal hydrophobic packing around three Val residues in its central CORE domain, which are replaced with Ile residues in D. rerio AK (AKDr). The Val-to-Ile mutations that improve hydrophobic CORE packing in AKNc increased stability at high temperatures but decreased activity at low temperatures, suggesting that the suboptimal hydrophobic CORE packing is important for cold adaptation. Such linkage between stability and activity was also observed in AKDr. Ile-to-Val mutations that destabilized the tropical AK resulted in increased activity at low temperatures. Our results provide the structural basis of cold adaptation of a psychrophilic enzyme from a multicellular, eukaryotic organism, and highlight the similarities and differences in the structural adjustment of vertebrate and bacterial psychrophilic AKs during cold adaptation.

A novel calcium-binding peptide from Antarctic krill protein hydrolysates and identification of binding sites of calcium-peptide complex

Trypsin was used for preparing peptides with high calcium-binding capacity from Antarctic krill. Hydroxyapatite chromatography (HAC), size-exclusion chromatography (SEC), and reversed phase high performance liquid chromatography (RP-HPLC) were used to capture and purify calcium-binding peptides. The peptide sequence was determined to be VLGYIQIR (N- to C-terminal, MW = 960.58 Da), using LTQ Orbitrap XL. According to the results of FTIR and mass spectrometry, chelating site of calcium ions may possibly involve the carbonal or amino groups of Gln, Ile and Arg residues. Molecular dynamic simulation showed the conformation of peptide was markedly varied, and the distance between calcium ion and Gln and Ile residues was changing all the time. However, the distance between calcium ion and carboxyl oxygen of arginine residues was not changed significantly from 2 ns to 100 ns. Identified peptide can be used as a novel calcium supplement.

The Phe-Ile Zipper: A Specific Interaction Motif Drives Antiparallel Coiled-Coil Hexamer Formation.

Coiled coils are a robust motif for exploring amino acid interactions, generating unique supramolecular structures, and expanding the functional properties of biological materials. A recently discovered antiparallel coiled-coil hexamer (ACC-Hex, peptide 1) exhibits a unique interaction in which Phe and Ile residues from adjacent α-helices interact to form a Phe-Ile zipper within the hydrophobic core. Analysis of the X-ray crystallographic structure of ACC-Hex suggests that the stability of the six-helix bundle relies on specific interactions between the Phe and Ile residues. The Phe-Ile zipper is unprecedented and represents a powerful tool for utilizing the Phe-Ile interactions to direct supramolecular assembly. To further probe and understand the limits of the Phe-Ile zipper, we designed peptide sequences with natural and unnatural amino acids placed at the Phe and Ile residue positions. Using size exclusion chromatography and small-angle X-ray scattering, we found that the proper assembly of ACC-Hex from monomers is sensitive to subtle changes in side chain steric bulk and hydrophobicity introduced by mutations at the Phe and Ile residue positions. Of the sequence variants that formed ACC-Hex, mutations in the hydrophobic core significantly affected the stability of the hexamer, from a ΔGuw of 2-8 kcal mol-1. Additional sequences were designed to further probe and enhance the stability of the ACC-Hex system by maximizing salt bridging between the solvent-exposed residues. Finally, we expanded on the generality of the Phe-Ile zipper, creating a unique sequence that forms an antiparallel hexamer that is topologically similar to ACC-Hex but atomistically unique.

Effect of Storage on Lactase-Treated β-Casein and β-Lactoglobulin with Respect to Bitter Peptide Formation and Subsequent in Vitro Digestibility.

Using active lactose to hydrolyze lactose during storage is a common process to produce lactose-hydrolyzed (LH) milk. Proteolysis induced by residual proteases in commercial lactase was studied in a system using purified β-casein or β-lactoglobulin during a 60-day storage period at 22 or 38 °C. The proteolysis of β-casein by residual proteases occurred more extensively than that of β-lactoglobulin. Peptidomic analysis by LC-ESI-MS/MS revealed that Ile, Leu, Tyr, and Phe residues near the C-terminus of β-casein were the main sites of cleavage by the residual proteases, generating assumed bitter peptides. In the subsequent in vitro digestion study, proteolysis during storage was shown to greatly affect the subsequent digestibility of β-casein, leading to an elevated degree of hydrolysis and the formation of new digested peptides. This study highlights the potential influence of residual proteases in commercial lactase on the storage quality and digestibility of LH milk containing active lactase.

Reinvestigation of the biological activity of d-allo-ShK protein

ShK toxin from the sea anemone Stichodactyla helianthus is a 35-residue protein that binds to the Kv1.3 ion channel with high affinity. Recently we determined the X-ray structure of ShK toxin by racemic crystallography, in the course of which we discovered that d-ShK has a near-background IC50 value ∼50,000 times lower than that of the l-ShK toxin. This lack of activity was at odds with previously reported results for an ShK diastereomer designated d-allo-ShK, for which significant biological activity had been observed in a similar receptor-blocking assay. As reported, d-allo-ShK was made up of d-amino acids, but with retention of the natural stereochemistry of the chiral side chains of the Ile and Thr residues, i.e. containing d-allo-Ile and d-allo-Thr along with d-amino acids and glycine. To understand its apparent biological activity, we set out to chemically synthesize d-allo-ShK and determine its X-ray structure by racemic crystallography. Using validated allo-Thr and allo-Ile, both l-allo-ShK and d-allo-ShK polypeptide chains were prepared by total chemical synthesis. Neither the l-allo-ShK nor the d-allo-ShK polypeptides folded, whereas both l-ShK and d-ShK folded smoothly under the same conditions. Re-examination of NMR spectra of the previously reported d-allo-ShK protein revealed that diagnostic Thr and Ile signals were the same as for authentic d-ShK. On the basis of these results, we conclude that the previously reported d-allo-ShK was in fact d-ShK, the true enantiomer of natural l-ShK toxin, and that the apparent biological activity may have arisen from inadvertent contamination with trace amounts of l-ShK toxin.

Flipping states: a few key residues decide the winning conformation of the only universally conserved transcription factor.

Transcription factors from the NusG family bind to the elongating RNA polymerase to enable synthesis of long RNAs in all domains of life. In bacteria, NusG frequently co-exists with specialized paralogs that regulate expression of a small set of targets, many of which encode virulence factors. Escherichia coli RfaH is the exemplar of this regulatory mechanism. In contrast to NusG, which freely binds to RNA polymerase, RfaH exists in a structurally distinct autoinhibitory state in which the RNA polymerase-binding site is buried at the interface between two RfaH domains. Binding to an ops DNA sequence triggers structural transformation wherein the domains dissociate and RfaH refolds into a NusG-like structure. Formation of the autoinhibitory state, and thus sequence-specific recruitment, represents the decisive step in the evolutionary history of the RfaH subfamily. We used computational and experimental approaches to identify the residues that confer the unique regulatory properties of RfaH. Our analysis highlighted highly conserved Ile and Phe residues at the RfaH interdomain interface. Replacement of these residues with equally conserved Glu and Val counterpart residues in NusG destabilized interactions between the RfaH domains and allowed sequence-independent recruitment to RNA polymerase, suggesting a plausible pathway for diversification of NusG paralogs.

Disruption of drug-resistant biofilms using de novo designed short α-helical antimicrobial peptides with idealized facial amphiphilicity.

Antimicrobial peptides (AMPs) are increasingly explored as therapeutics for drug-resistant and biofilm-related infections to help expand the size and quality of the current antibiotic pipeline in the face of mounting antimicrobial resistance. Here, synthetic peptides rationally designed based upon principles governing the folding of natural α-helical AMPs, comprising the backbone sequence (X1Y1Y2X2)n, and which assemble into α-helical structures with idealized facial amphiphilicity, is presented. These multifunctional peptide amphiphiles demonstrate high bacterial selectivity, promote the disruption of pre-formed drug-resistant biofilms, and effectively neutralize endotoxins at low micromolar concentrations. Overall, the design strategies presented here could provide a useful tool for developing therapeutic peptides with broad-ranging clinical applications from the treatment and prevention of drug-resistant biofilms to the neutralization of bacterial endotoxins.

Mutations near the cleavage site of enterocin NKR-5-3B prepeptide reveal new insights into its biosynthesis.

Enterocin NKR-5-3B (Ent53B) is a 64-residue novel circular bacteriocin synthesized from an 87-residue prepeptide. Albeit through a still unknown mechanism, the EnkB1234 biosynthetic enzyme complex processes the prepeptide to yield its mature active, circular form. To gain insights into the key region/residue that plays a role in Ent53 maturation, several mutations near the cleavage site on the precursor peptide were generated. The interaction of the precursor peptide and EnkB1234 appeared to be hydrophobic in nature. At the Leu1 position, only mutations with helix structure-promoting hydrophobic residues (Ala, Ile, Val or Phe) were able to yield the mature Ent53B derivative. In this study, we also highlight the possible conformation-stabilizing role of the Ent53B leader peptide on the precursor peptide for its interaction with its biosynthetic enzyme complex. Any truncations of the leader peptide moiety interfered in the processing of the prepeptide. However, when propeptides of other circular bacteriocins (circularin A, leucocyclicin Q or lactocyclicin Q) were cloned at the C-terminus of the leader peptide, EnkB1234 could not process them to yield a mature bacteriocin. Taken together, these findings offer new perspectives in our understanding of the possible molecular mechanism of the biosynthesis of this circular bacteriocin. These new perspectives will help advance our current understanding to eventually elucidate circular bacteriocin biosynthesis. Understanding the biosynthetic mechanism of circular bacteriocins will materialize their application potential.

The Michaelis Complex of Arginine Kinase Samples the Transition State at a Frequency That Matches the Catalytic Rate.

Arginine kinase (AK), which is a member of the phosphagen kinase family, serves as a model system for studying the structural and dynamic determinants of biomolecular enzyme catalysis of all major states involved of the enzymatic cycle. These states are the apo state (substrate free), the Michaelis complex analogue AK:Arg:Mg·AMPPNP (MCA), a product complex analogue AK:pAIE:Mg·ADP (PCA), and the transition state analogue AK:Arg:Mg·ADP:NO3- (TSA). The conformational dynamics of these states have been studied by NMR relaxation dispersion measurements of the methyl groups of the Ile, Leu, and Val residues at two static magnetic fields. Although all states undergo significant amounts of μs-ms time scale dynamics, only the MCA samples a dominant excited state that resembles the TSA, as evidenced by the strong correlation between the relaxation dispersion derived chemical shift differences Δω and the equilibrium chemical shift differences Δδ of these states. The average lifetime of the MCA is 36 ms and the free energy difference to the TSA-like form is 8.5 kJ/mol. It is shown that the conformational energy landscape of the Michaelis complex analogue is shaped in a way that at room temperature it channels passage to the transition state, thereby determining the rate-limiting step of the phosphorylation reaction of arginine. Conversely, relaxation dispersion experiments of the TSA reveal that it samples the structures of the Michaelis complex analogue or the apo state as its dominant excited state. This reciprocal behavior shows that the free energy of the TSA, with all ligands bound, is lower by only about 8.9 kJ/mol than that of the Michaelis or apo complex conformations with the TSA ligands present.

Positional Scanning Identifies the Molecular Determinants of a High Affinity Multi-Leucine Inhibitor for Furin and PACE4

The proprotein convertase family of enzymes includes seven endoproteases with significant redundancy in their cleavage activity. We previously described the peptide Ac-LLLLRVK-Amba that displays potent inhibitory effects on both PACE4 and prostate cancer cell lines proliferation. Herein, the molecular determinants for PACE4 and furin inhibition were investigated by positional scanning using peptide libraries that substituted its leucine core with each natural amino acid. We determined that the incorporation of basic amino acids led to analogues with improved inhibitory potency toward both enzymes, whereas negatively charged residues significantly reduced it. All the remaining amino acids were in general well tolerated, with the exemption of the P6 position. However, not all of the potent PACE4 inhibitors displayed antiproliferative activity. The best analogues were obtained by the incorporation of the Ile residue at the P5 and P6 positions. These substitutions led to inhibitors with increased PACE4 selectivity and potent antiproliferative effects.

Left or Right: How Does Amino Acid Chirality Affect the Handedness of Nanostructures Self-Assembled from Short Amphiphilic Peptides?

Peptide and protein fibrils have attracted an enormous amount of interests due to their relevance to many neurodegenerative diseases and their potential applications in nanotechnology. Although twisted fibrils are regarded as the key intermediate structures of thick fibrils or bundles of fibrils, the factors determining their twisting tendency and their handedness development from the molecular to the supramolecular level are still poorly understood. In this study, we have designed three pairs of enantiomeric short amphiphilic peptides: LI3LK and DI3DK, LI3DK and DI3LK, and LaI3LK and DaI3DK, and investigated the chirality of their self-assembled nanofibrils through the combined use of atomic force microscopy (AFM), circular dichroism (CD) spectroscopy, scanning electron microscopy (SEM), and molecular dynamic (MD) simulations. The results indicated that the twisted handedness of the supramolecular nanofibrils was dictated by the chirality of the hydrophilic Lys head at the C-terminal, while their characteristic CD signals were determined by the chirality of hydrophobic Ile residues. MD simulations delineated the handedness development from molecular chirality to supramolecular handedness by showing that the β-sheets formed by LI3LK, LaI3LK, and DI3LK exhibited a propensity to twist in a left-handed direction, while the ones of DI3DK, DaI3DK, and LI3DK in a right-handed twisting orientation.

Inversion of the Side-Chain Stereochemistry of Indvidual Thr or Ile Residues in a Protein Molecule: Impact on the Folding, Stability, and Structure of the ShK Toxin.

ShK toxin is a cysteine-rich 35-residue protein ion-channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side-chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo-Thr or an allo-Ile residue. The three allo-Thr or allo-Ile-containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi-racemic X-ray crystallography and were similar to wild-type ShK. All three ShK analogues retained ion-channel blocking activity.

Inversion of the Side‐Chain Stereochemistry of Indvidual Thr or Ile Residues in a Protein Molecule: Impact on the Folding, Stability, and Structure of the ShK Toxin

AbstractShK toxin is a cysteine‐rich 35‐residue protein ion‐channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side‐chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo‐Thr or an allo‐Ile residue. The three allo‐Thr or allo‐Ile‐containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi‐racemic X‐ray crystallography and were similar to wild‐type ShK. All three ShK analogues retained ion‐channel blocking activity.

Antigenotoxicity Activity of Papaya (Carica papaya L.) Leaf Ethanolic Extract on Swiss Mice Induced Cyclophosphamide through Mammalian In Vivo Micronucleus Test

Cyclophosphamide (CPA) is an effective chemotherapeutic agent, but has side effect, causing DNA damage (genotoxic). Papaya leaf (Carica papaya L.) is known has flavonoid compound, quercetin. Quercetin is known has DNA protecting effect (antigenotoxic effect) by metabolism modulation. Thus, the aim of this research is to investigate the antigenotoxic effect of ethanolic extract of papaya (Carica papaya L.) leaf (EEPL) on CPA induced mice. The antigenotoxic effect was evaluated by mammalian in vivo micronucleus test. EEPL was orally administered as single treatment at dose 1000 mg/kgBW and in combination with CPA 50 mg/kgBW at dose 250 mg/kgBW; 500 mg/kgBW; and 1000 mg/kgBW. Molecular docking using PLANTS on CYP 3A4 was performed to explore the antigenotoxic effect mechanism. The three different combination dose of EEPL with CPA significantly (P&lt;0.05) decreased the amount of micronucleated polychromatic erytrhocyte (MNPCE)/1000 polychromatic erythrocyte (PCE) and also increased % PCE/(PCE+normochromatic erythrocyte (NCE)), compared with single dose of CPA. Nevertheless, the antigenotoxic effect wasn’t significant compared with each combination dose. The docking score result showed quercetin (-82,41) has more potent interaction to CYP 3A4 than cyclophosphamide (-70,16) and both of them has similar active site at amino acid residue Ile 369 and Thr 309. The results obtained indicated that EEPL at dose 250 mg/KgBB is the optimal dose as antigenotoxic agent by interaction between quercetin with CYP 3A4 based on molecular docking.Keywords: antigenotoxic, Carica papaya L., MNPCE, in vivo