Cysteine Oxidation in Human Galectin-1 Occurs Sequentially via a Folded Intermediate to a Fully Oxidized Unfolded Form.

Galectins are multifunctional effectors in cellular homeostasis and dysregulation. Oxidation of human galectin-1 (Gal-1) with its six sulfhydryls produces a disulfide-bridged oxidized form that lacks normal lectin activity yet gains new glycan-independent functionality. Nevertheless, the mechanistic details as to how Gal-1 oxidation occurs remain unclear. Here, we used 15N and 13C HSQC NMR spectroscopy to gain structural insight into the CuSO4-mediated path of Gal-1 oxidation and identified a minimum two-stage conversion process. During the first phase, disulfide bridges form slowly between C16-C88 and/or C42-C66 to produce a partially oxidized, conformationally flexible intermediate that retains the ability to bind lactose. Site-directed mutagenesis of C16 to S16 impedes the onset of this overall slow process. During the second phase, increased motional dynamics of the intermediate enable the relatively distant C2 and C130 residues to form the third and final disulfide bond, leading to an unfolded state and consequent dimer dissociation. This fully oxidized end state loses the ability to bind lactose, as shown by the hemagglutination assay. Consistent with this model, we observed that the Gal-1 C2S mutant maintains intermediate-state structural features with a free sulfhydryl group at C130. Incubation with dithiothreitol reduces all disulfide bonds and allows the lectin to revert to its native state. Thus, the sequential, non-random formation of three disulfide bridges in Gal-1 in an oxidative environment acts as a molecular switch for fundamental changes to its functionality. These data inspire detailed bioactivity analysis of the structurally defined oxidized intermediate in, e.g., acute and chronic inflammation.

Effect of magnetic field-assisted fermentation on the in vitro protein digestibility and molecular structure of rapeseed meal.

BACKGROUND: There has been a significant growth in demand for plant-derived protein, and this has been accompanied by an increasing need for sustainable animal-feed options. The aim of this study was to investigate the effect of magnetic field-assisted solid fermentation (MSSF) on the in vitro protein digestibility (IVPD) and functional and structural characteristics of rapeseed meal (RSM) with a mutant strain of Bacillus subtilis. RESULTS: Our investigation demonstrated that the MSSF nitrogen release rate reached 86.3% after 96 h of fermentation. The soluble protein and peptide content in magnetic field feremented rapeseed meal reached 29.34 and 34.49 mg mL-1 after simulated gastric digestion, and the content of soluble protein and peptide in MF-FRSM reached 61.81 and 69.85 mg mL-1 after simulated gastrointestinal digestion, which significantly increased (p > 0.05) compared with the fermented rapeseed meal (FRSM). Studies of different microstructures - using scanning electron microscopy (SEM) and atomic force microscopy (AFM) - and protein secondary structures have shown that the decline in intermolecular or intramolecular cross-linking leads to the relative dispersion of proteins and improves the rate of nitrogen release. The smaller number of disulfide bonds and conformational alterations suggests that the IVPD of RSM was improved. CONCLUSIONS: Magnetic field-assisted solid fermentation can be applied to enhance the nutritional and protein digestibility of FRSM. © 2024 Society of Chemical Industry.

Reverse Chemical Ecology Suggests Putative Primate Pheromones.

Pheromonal communication is widespread among living organisms, but in apes and particularly in humans there is currently no strong evidence for such phenomenon. Among primates, lemurs use pheromones to communicate within members of the same species, whereas in some monkeys such capabilities seem to be lost. Chemical communication in humans appears to be impaired by the lack or malfunctioning of biochemical tools and anatomical structures mediating detection of pheromones. Here, we report on a pheromone-carrier protein (SAL) adopting a "reverse chemical ecology" approach to get insights on the structures of potential pheromones in a representative species of lemurs (Microcebus murinus) known to use pheromones, Old-World monkeys (Cercocebus atys) for which chemical communication has been observed, and humans (Homo sapiens), where pheromones and chemical communication are still questioned. We have expressed the SAL orthologous proteins of these primate species, after reconstructing the gene encoding the human SAL, which is disrupted due to a single base mutation preventing its translation into RNA. Ligand-binding experiments with the recombinant SALs revealed macrocyclic ketones and lactones as the best ligands for all three proteins, suggesting cyclopentadecanone, pentadecanolide, and closely related compounds as the best candidates for potential pheromones. Such hypothesis agrees with the presence of a chemical very similar to hexadecanolide in the gland secretions of Mandrillus sphinx, a species closely related to C. atys. Our results indicate that the function of this carrier protein has not changed much during evolution from lemurs to humans, although its physiological role has been certainly impaired in humans.

Epipolythiodioxopiperazine-Based Natural Products: Building Blocks, Biosynthesis and Biological Activities.

Epipolythiodioxopiperazines (ETPs) are fungal secondary metabolites that share a 2,5-diketopiperazine scaffold built from two amino acids and bridged by a sulfide moiety. Modifications of the core and the amino acid side chains, for example by methylations, acetylations, hydroxylations, prenylations, halogenations, cyclizations, and truncations create the structural diversity of ETPs and contribute to their biological activity. However, the key feature responsible for the bioactivities of ETPs is their sulfide moiety. Over the last years, combinations of genome mining, reverse genetics, metabolomics, biochemistry, and structural biology deciphered principles of ETP production. Sulfurization via glutathione and uncovering of the thiols followed by either oxidation or methylation crystallized as fundamental steps that impact expression of the biosynthesis cluster, toxicity and secretion of the metabolite as well as self-tolerance of the producer. This article showcases structure and activity of prototype ETPs such as gliotoxin and discusses the current knowledge on the biosynthesis routes of these exceptional natural products.

Divalent cations influence the dimerization mode of murine S100A9 protein by modulating its disulfide bond pattern.

S100A9, with its congener S100A8, belongs to the S100 family of calcium-binding proteins found exclusively in vertebrates. These two proteins are major constituents of neutrophils. In response to a pathological condition, they can be released extracellularly and become alarmins that induce both pro- and anti-inflammatory signals, through specific cell surface receptors. They also act as antimicrobial agents, mainly as a S100A8/A9 heterocomplex, through metal sequestration. The mechanisms whereby divalent cations modulate the extracellular functions of S100A8 and S100A9 are still unclear. Importantly, it has been proposed that these ions may affect both the ternary and quaternary structure of these proteins, thereby influencing their physiological properties. In the present study, we report the crystal structures of WT and C80A murine S100A9 (mS100A9), determined at 1.45 and 2.35 Å resolution, respectively, in the presence of calcium and zinc. These structures reveal a canonical homodimeric form for the protein. They also unravel an intramolecular disulfide bridge that stabilizes the C-terminal tail in a rigid conformation, thus shaping a second Zn-binding site per S100A9 protomer. In solution, mS100A9 apparently binds only two zinc ions per homodimer, with an affinity in the micromolar range, and aggregates in the presence of excess zinc. Using mass spectrometry, we demonstrate that mS100A9 can form both non-covalent and covalent homodimers with distinct disulfide bond patterns. Interestingly, calcium and zinc seem to affect differentially the relative proportion of these forms. We discuss how the metal-dependent interconversion between mS100A9 homodimers may explain the versatility of physiological functions attributed to the protein.

The Odorant-Binding Proteins of the Spider Mite Tetranychus urticae.

Spider mites are one of the major agricultural pests, feeding on a large variety of plants. As a contribution to understanding chemical communication in these arthropods, we have characterized a recently discovered class of odorant-binding proteins (OBPs) in Tetranychus urticae. As in other species of Chelicerata, the four OBPs of T. urticae contain six conserved cysteines paired in a pattern (C1-C6, C2-C3, C4-C5) differing from that of insect counterparts (C1-C3, C2-C5, C4-C6). Proteomic analysis uncovered a second family of OBPs, including twelve members that are likely to be unique to T. urticae. A three-dimensional model of TurtOBP1, built on the recent X-ray structure of Varroa destructor OBP1, shows protein folding different from that of insect OBPs, although with some common features. Ligand-binding experiments indicated some affinity to coniferyl aldehyde, but specific ligands may still need to be found among very large molecules, as suggested by the size of the binding pocket.

Breaking a Couple: Disulfide Reducing Agents.

Cysteine is present in a large number of natural and synthetic (bio)molecules. Although the thiol side chain of Cys can be in a free form, in most cases it forms a disulfide bond either with a second Cys (bridge) or with another thiol, as in the case of protecting groups. Efficient reduction of these disulfide bridges is a requirement for many applications of Cys-containing molecules in the fields of chemistry and biochemistry. Here we review reducing methods for disulfide bonds, taking into consideration the solubility of the substrates when selecting the appropriate reducing reagent.

Recent Chemical Approaches for Site-Specific Conjugation of Native Antibodies: Technologies toward Next-Generation Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs), which consist of three components, antibody, linker, and payload, can function as "magic bullets". These conjugates offer the ability to target drug delivery to specific cells, based on cell-specific recognition and the binding of an antigen by a monoclonal antibody (mAb). In particular, by delivering a cytotoxic payload to cancer cells, ADCs are expected to provide a breakthrough in oncology treatments by providing a way to increase efficacy and decrease toxicity, in comparison with traditional chemotherapeutic treatments. The development of ADC therapeutics has dramatically progressed in the past decade and two ADCs have been approved and used as anticancer drugs in the clinic. However, several critical issues regarding the performance of ADCs are still being discussed and investigated. Indeed, in the past few years, several groups have reported that, changing the number and position of the drug payloads in the ADCs, affects the pharmacokinetics, drug release rates, and biological activity. The use of conventional heterogeneous conjugation methods for ADC preparation results in the drug/antibody ratio and connecting position of the payload having stochastic distributions. Therefore, it is important to investigate how these potential problems can be circumvented through site-specific conjugation. Herein, various site-specific chemical conjugation strategies with native mAbs that are currently used for the production of ADCs, including residue-selective labeling for generating ADCs, disulfide rebridging, and affinity-peptide-mediated site-specific chemical conjugation technologies, are reviewed and described.

Dimeric C34 Derivatives Linked through Disulfide Bridges as New HIV-1 Fusion Inhibitors.

C34, a 34-mer fragment peptide, is contained in the HIV-1 envelope protein gp41. A dimeric derivative of C34 linked through a disulfide bridge at its C terminus was synthesized and found to display potent anti-HIV activity, comparable with that of a previously reported PEGylated dimer of C34REG. The reduction in the size of the linker moiety for dimerization was thus successful, and this result might shed some light on the mechanism of the suppression of six-helix bundle formation by these C34 dimeric derivatives. Addition of a Gly-Cys(CH2 CONH2 )-Gly-Gly motif at the N-terminal position of a C34 monomeric derivative significantly increased the anti-HIV-1 activity. This moiety functions as a new pharmacophore, and this might provide a useful insight into the design of potent HIV-1 fusion inhibitors.

Mass Spectrometry- and Computational Structural Biology-Based Investigation of Proteins and Peptides.

Recent developments of mass spectrometry (MS) allow us to identify, estimate, and characterize proteins and protein complexes. At the same time, structural biology helps to determine the protein structure and its structure-function relationship. Together, they aid to understand the protein structure, property, function, protein-complex assembly, protein-protein interaction, and dynamics. The present chapter is organized with illustrative results to demonstrate how experimental mass spectrometry can be combined with computational structural biology for detailed studies of protein's structures. We have used tumor differentiation factor protein/peptide as ligand and Hsp70/Hsp90 as receptor protein as examples to study ligand-protein interaction. To investigate possible protein conformation, we will describe two proteins-lysozyme and myoglobin. As an application of MS-based assignment of disulfide bridges, the case of the spider venom polypeptide Phα1ß will also be discussed.

Disulfide Bonds Enable Accelerated Protein Evolution.

The different proteins of any proteome evolve at enormously different rates. What factors contribute to this variability, and to what extent, is still a largely open question. We hypothesized that disulfide bonds, by increasing protein stability, should make proteins' structures relatively independent of their amino acid sequences, thus acting as buffers of deleterious mutations and enabling accelerated sequence evolution. In agreement with this hypothesis, we observed that membrane proteins with disulfide bonds evolved 88% faster than those without disulfide bonds, and that extracellular proteins with disulfide bonds evolved 49% faster than those without disulfide bonds. In addition, genes encoding proteins with disulfide bonds exhibit an increased likelihood of showing signatures of positive selection. Multivariate analyses indicate that the trend is independent of a number of potentially confounding factors. The effect, however, is not observed among the longest proteins, which can become stabilized by mechanisms other than disulfide bonds.

Crosslinking Between Trichocyte Keratins and Keratin Associated Proteins.

Trichocyte keratins differ considerably from their epithelial cousins in having a higher number of cysteine residues, of which the greater proportion are located in the head and tail regions of these proteins. Coupled with this is the presence of a large number of keratin associated proteins in these fibres that are high in their cysteine content, the high sulfur proteins and ultra-high sulfur proteins. Thus it is the crosslinking that occurs between the cysteines in the keratins and KAPs that is an important determinant in the functionality of wool and hair fibres. Studies have shown the majority of the cysteine residues are involved in internal crosslinking in the KAPs leaving only a few specific cysteines to interact with the keratins, with most evidence pointing to interactions between these KAP cysteines and the keratin head groups.

Photochemical and Structural Studies on Cyclic Peptide Models.

Ultra-violet (UV) irradiation has a significant impact on the structure and function of proteins that is supposed to be in relationship with the tryptophan-mediated photolysis of disulfide bonds. To investigate the correlation between the photoexcitation of Trp residues in polypeptides and the associated reduction of disulfide bridges, a series of small, cyclic oligopeptide models were analyzed in this work. Average distances between the aromatic side chains and the disulfide bridge were determined following molecular mechanics (MM) geometry optimizations. In this way, the possibility of cation⁻π interactions was also investigated. Molecular mechanics calculations revealed that the shortest distance between the side chain of the Trp residues and the disulfide bridge is approximately 5 Å in the cyclic pentapeptide models. Based on this, three tryptophan-containing cyclopeptide models were synthesized and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Experimental data and detailed molecular dynamics (MD) simulations were in good agreement with MM geometry calculations. Selected model peptides were subjected to photolytic degradation to study the correlation of structural features and the photolytic cleavage of disulfide bonds in solution. Formation of free sulfhydryl groups upon illumination with near UV light was monitored by fluorescence spectroscopy after chemical derivatization with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) and mass spectrometry. Liquid cromatography-mass spectrometry (LC-MS) measurements indicated the presence of multiple photooxidation products (e.g., dimers, multimers and other oxidated products), suggesting that besides the photolysis of disulfide bonds secondary photolytic processes take place.

Novel synthetic analogues of avian ß-defensin-12: the role of charge, hydrophobicity, and disulfide bridges in biological functions.

BACKGROUND: Avian ß-defensins (AvBD) possess broad-spectrum antimicrobial, LPS neutralizing and chemotactic properties. AvBD-12 is a chemoattractant for avian immune cells and mammalian dendritic cells (JAWSII) - a unique feature that is relevant to the applications of AvBDs as chemotherapeutic agents in mammalian hosts. To identify the structural components essential to various biological functions, we have designed and evaluated seven AvBD analogues. RESULTS: In the first group of analogues, the three conserved disulfide bridges were eliminated by replacing cysteines with alanine and serine residues, peptide hydrophobicity and charge were increased by changing negatively charged amino acid residues to hydrophobic (AvBD-12A1) or positively charged residues (AvBD-12A2 and AvBD-12A3). All three analogues in this group showed improved antimicrobial activity, though AvBD-12A3, with a net positive charge of +9, hydrophobicity of 40% and a predicted CCR2 binding domain, was the most potent antimicrobial peptide. AvBD-12A3 also retained more than 50% of wild type chemotactic activity. In the second group of analogues (AvBD-12A4 to AvBD-12A6), one to three disulfide bridges were removed via substitution of cysteines with isosteric amino acids. Their antimicrobial activity was compromised and chemotactic activity abolished. The third type of analogue was a hybrid that had the backbone of AvBD-12 and positively charged amino acid residues AvBD-6. The antimicrobial and chemotactic activities of the hybrid resembled that of AvBD-6 and AvBD-12, respectively. CONCLUSIONS: While the net positive charge and charge distribution have a dominating effect on the antimicrobial potency of AvBDs, the three conserved disulfide bridges are essential to the chemotactic property and the maximum antimicrobial activity. Analogue AvBD-12A3 with a high net positive charge, a moderate degree of hydrophobicity and a CCR2-binding domain can serve as a template for the design of novel antimicrobial peptides with chemotactic property and salt resistance.

Impurity determination for hepcidin by liquid chromatography-high resolution and ion mobility mass spectrometry for the value assignment of candidate primary calibrators.

In metrology institutes, the state-of-the-art for purity analysis of peptides/proteins mainly addresses short and unfolded peptides. Important developments are anticipated for the characterization of nonlinear peptides or proteins. Hepcidin 1-25 is an interesting model system because this small protein contains four disulfide bridges with a particular connectivity that is difficult to reproduce and could induce a bias in quantification. Hepcidin 1-25 is involved in iron-related disorders and anemia, in an inflammatory context, and its clinical relevance in neurodegenerative disorders is under investigation. It is also an emerging biomarker. Recent inter-laboratory studies showed a need for standardization of hepcidin assay and the need to produce certified reference materials. This paper discusses two hepcidin standards from different synthesis pathways that have been characterized by high-resolution mass spectrometry and ion mobility mass spectrometry.

Multiple disulfide bridges modulate conformational stability and flexibility in hyperthermophilic archaeal purine nucleoside phosphorylase.

5'-Deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus is a hexameric hyperthermophilic protein containing in each subunit two pairs of disulfide bridges, a CXC motif, and one free cysteine. The contribution of each disulfide bridge to the protein conformational stability and flexibility has been assessed by comparing the thermal unfolding and the limited proteolysis of the wild-type enzyme and its variants obtained by site-directed mutagenesis of the seven cysteine residues. All variants catalyzed efficiently MTA cleavage with specific activity similar to the wild-type enzyme. The elimination of all cysteine residues caused a substantial decrease of ΔHcal (850 kcal/mol) and Tmax (39°C) with respect to the wild-type indicating that all cysteine pairs and especially the CXC motif significantly contribute to the enzyme thermal stability. Disulfide bond Cys200-Cys262 and the CXC motif weakly affected protein flexibility while the elimination of the disulfide bond Cys138-Cys205 lead to an increased protease susceptibility. Experimental evidence from limited proteolysis, differential scanning calorimetry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions also allowed to propose a stabilizing role for the free Cys164.

Structure-function analysis of Avian ß-defensin-6 and ß-defensin-12: role of charge and disulfide bridges.

BACKGROUND: Avian beta-defensins (AvBD) are small, cationic, antimicrobial peptides. The potential application of AvBDs as alternatives to antibiotics has been the subject of interest. However, the mechanisms of action remain to be fully understood. The present study characterized the structure-function relationship of AvBD-6 and AvBD-12, two peptides with different net positive charges, similar hydrophobicity and distinct tissue expression profiles. RESULTS: AvBD-6 was more potent than AvBD-12 against E. coli, S. Typhimurium, and S. aureus as well as clinical isolates of extended spectrum beta lactamase (ESBL)-positive E. coli and K. pneumoniae. AvBD-6 was more effective than AvBD-12 in neutralizing LPS and interacting with bacterial genomic DNA. Increasing bacterial concentration from 10(5) CFU/ml to 10(9) CFU/ml abolished AvBDs' antimicrobial activity. Increasing NaCl concentration significantly inhibited AvBDs' antimicrobial activity, but not the LPS-neutralizing function. Both AvBDs were mildly chemotactic for chicken macrophages and strongly chemotactic for CHO-K1 cells expressing chicken chemokine receptor 2 (CCR2). AvBD-12 at higher concentrations also induced chemotactic migration of murine immature dendritic cells (DCs). Disruption of disulfide bridges abolished AvBDs' chemotactic activity. Neither AvBDs was toxic to CHO-K1, macrophages, or DCs. CONCLUSIONS: AvBDs are potent antimicrobial peptides under low-salt conditions, effective LPS-neutralizing agents, and broad-spectrum chemoattractant peptides. Their antimicrobial activity is positively correlated with the peptides' net positive charges, inversely correlated with NaCl concentration and bacterial concentration, and minimally dependent on intramolecular disulfide bridges. In contrast, their chemotactic property requires the presence of intramolecular disulfide bridges. Data from the present study provide a theoretical basis for the design of AvBD-based therapeutic and immunomodulatory agents.

Oxidation of the N-terminal domain of the wheat metallothionein Ec -1 leads to the formation of three distinct disulfide bridges.

Metallothioneins (MTs) are low molecular weight proteins, characterized by a high cysteine content and the ability to coordinate large amounts of d(10) metal ions, for example, Zn(II), Cd(II), and Cu(I), in form of metal-thiolate clusters. Depending on intracellular conditions such as redox potential or metal ion concentrations, MTs can occur in various states ranging from the fully metal-loaded holo- to the metal-free apo-form. The Cys thiolate groups in the apo-form can be either reduced or be involved in disulfide bridges. Although oxidation-mediated Zn(II) release might be a possible mechanism for the regulation of Zn(II) availability by MTs, no concise information regarding the associated pathways and the structure of oxidized apo-MT forms is available. Using the well-studied Zn2 γ-Ec -1 domain of the wheat Zn6 Ec -1 MT we attempt here to answer several question regarding the structure and biophysical properties of oxidized MT forms, such as: (1) does disulfide bond formation increase the stability against proteolysis, (2) is the overall peptide backbone fold similar for the holo- and the oxidized apo-MT form, and (3) are disulfide bridges specifically or randomly formed? Our investigations show that oxidation leads to three distinct disulfide bridges independently of the applied oxidation conditions and of the initial species used for oxidation, that is, the apo- or the holo-form. In addition, the oxidized apo-form is as stable against proteolysis as Zn2 γ-Ec -1, rendering the currently assumed degradation of oxidized MTs unlikely and suggesting a role of the oxidation process for the extension of protein lifetime in absence of sufficient amounts of metal ions. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 295-308, 2016.

Two Pseudopolymorphic Star-Shaped Tetranuclear Co(3+) Compounds with Disulfide Anions Exhibiting Two Different Connection Modes and Promising Photocatalytic Properties.

The compound [Co4(C6H14N2)4(µ4-S2)2(µ2-S2)4] (I) and the pseudo-polymorph [Co4(C6H14N2)4(µ4-S2)2(µ2-S2)4]⋅4 H2O (II) were obtained under solvothermal conditions (C6H14N2=trans-1,2-diaminocyclohexane). The structures feature S2(2-) ions exhibiting two different coordination modes. Terminal S2(2-) entities join two Co(3+) centres in a µ2 fashion, whereas the central S2(2-) groups connect four Co(3+) cations in a µ4-coordination mode. Compound II can be transformed into compound I by heat and storage over P2O5 and storing compound I in humid air yields in the formation of compound II. The intermolecular interactions investigated through Hirshfeld surface analysis reveal that besides S⋅⋅⋅H bonding close contacts are associated with relatively weak H⋅⋅⋅H interactions. A detailed DFT analysis of the bonding situation explains the long S-S bonds in the µ4-bridging S2(2-) units and the short bonds for the S2(2-) moieties in the µ2-connecting mode. Photocatalytic hydrogen evolution experiments demonstrate the potential of compound II as catalyst.

A biotechnological approach for the production of new protein bioplastics.

The future of biomaterial production will leverage biotechnology based on the domestication of cells as biological factories. Plants, algae, and bacteria can produce low-environmental impact biopolymers. Here, two strategies were developed to produce a biopolymer derived from a bioengineered vacuolar storage protein of the common bean (phaseolin; PHSL). The cys-added PHSL* forms linear-structured biopolymers when expressed in the thylakoids of transplastomic tobacco leaves by exploiting the formation of inter-chain disulfide bridges. The same protein without signal peptide (ΔPHSL*) accumulates in Escherichia coli inclusion bodies as high-molar-mass species polymers that can subsequently be oxidized to form disulfide crosslinking bridges in order to increase the stiffness of the biomaterial, a valid alternative to the use of chemical crosslinkers. The E. coli cells produced 300 times more engineered PHSL, measured as percentage of total soluble proteins, than transplastomic tobacco plants. Moreover, the thiol groups of cysteine allow the site-specific PEGylation of ΔPHSL*, which is a desirable functionality in the design of a protein-based drug carrier. In conclusion, ΔPHSL* expressed in E. coli has the potential to become an innovative biopolymer.