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Thomas Scheibel - One of the best experts on this subject based on the ideXlab platform.
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Engineering of Silk Proteins for materials applications.
Current opinion in biotechnology, 2019Co-Authors: Merisa Saric, Thomas ScheibelAbstract:Silk combines biological properties, such as non-toxicity and biodegradability, with physico-chemical ones, for example, mechanical strength. Base on molecular engineering, nowadays also new non-Silk functions can be implemented in Silk materials. Driven by rational design and ingenuity, innovative recombinant Silk Proteins can be designed with a plethora of functions to address biomedical and technological challenges. Herein, we review advances in engineering Silk Proteins for tailored functions at the molecular level. Insights are provided in genetically engineered Silk fusions with functional or other structural Proteins and in hybrids with DNA. In such novel materials, self-assembly features of Silk are combined and utilized with expedient properties of the additional components. The availability of functionalized Silk materials is opening routes toward a whole set of novel applications not achievable with natural Silk or other polymers.
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Sequence Identification, Recombinant Production, and Analysis of the Self-Assembly of Egg Stalk Silk Proteins from Lacewing Chrysoperla carnea.
Biomolecules, 2017Co-Authors: Martin Neuenfeldt, Thomas ScheibelAbstract:Egg stalk Silks of the common green lacewing Chrysoperla carnea likely comprise at least three different Silk Proteins. Based on the natural spinning process, it was hypothesized that these Proteins self-assemble without shear stress, as adult lacewings do not use a spinneret. To examine this, the first sequence identification and determination of the gene expression profile of several Silk Proteins and various transcript variants thereof was conducted, and then the three major Proteins were recombinantly produced in Escherichia coli encoded by their native complementary DNA (cDNA) sequences. Circular dichroism measurements indicated that the Silk Proteins in aqueous solutions had a mainly intrinsically disordered structure. The largest Silk protein, which we named ChryC1, exhibited a lower critical solution temperature (LCST) behavior and self-assembled into fibers or film morphologies, depending on the conditions used. The second Silk protein, ChryC2, self-assembled into nanofibrils and subsequently formed hydrogels. Circular dichroism and Fourier transform infrared spectroscopy confirmed conformational changes of both Proteins into beta sheet rich structures upon assembly. ChryC3 did not self-assemble into any morphology under the tested conditions. Thereby, through this work, it could be shown that recombinant lacewing Silk Proteins can be produced and further used for studying the fiber formation of lacewing egg stalks.
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Recombinant Silk Production in Bacteria
Reference Module in Materials Science and Materials Engineering, 2016Co-Authors: David L Kaplan, Thomas ScheibelAbstract:Abstract Silks can be defined as secreted Proteins spun into fibrous structures. Characteristically, Silk Proteins are produced in specialized glands and stored in a fluid state in the lumen of the gland. As the fluid passes through the spinning duct, a rapid transformation to the solid state takes place and the Silk becomes water-insoluble. Silk fibers are diverse in function, depending on the biological source, such as spiders or Silkworms. The article describes the recent remarkable progress in understanding Silk genetics, structures and biophysics, as well as the recombinant production of Silk Proteins.
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processing of recombinant spider Silk Proteins into tailor made materials for biomaterials applications
Current Opinion in Biotechnology, 2014Co-Authors: Kristin Schacht, Thomas ScheibelAbstract:Spider Silk has extraordinary mechanical properties, is biocompatible and biodegradable, and therefore an ideal material for biomedical applications. However, a drawback for any application is the inhomogeneity of spider Silk, as seen for other natural materials, as well as the low availability due to the cannibalism of most spiders. Recently, developed recombinant spider Silk Proteins ensure constant material properties, as well as scalable production, and further the processing into morphologies other than fibres. Biotechnology enables genetic modification, broadening the range of applications, such as implant coatings, scaffolds for tissue engineering, wound dressing devices as well as drug delivery systems.
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coatings and films made of Silk Proteins
ACS Applied Materials & Interfaces, 2014Co-Authors: Christian B Borkner, Martina B Elsner, Thomas ScheibelAbstract:Silks are a class of proteinaceous materials produced by arthropods for various purposes. Spider dragline Silk is known for its outstanding mechanical properties, and it shows high biocompatibility, good biodegradability, and a lack of immunogenicity and allergenicity. The Silk produced by the mulberry Silkworm B. mori has been used as a textile fiber and in medical devices for a long time. Here, recent progress in the processing of different Silk materials into highly tailored isotropic and anisotropic coatings for biomedical applications such as tissue engineering, cell adhesion, and implant coatings as well as for optics and biosensors is reviewed.
David L Kaplan - One of the best experts on this subject based on the ideXlab platform.
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experimental methods for characterizing the secondary structure and thermal properties of Silk Proteins
Macromolecular Rapid Communications, 2019Co-Authors: Meghan Mcgill, Gregory P. Holland, David L KaplanAbstract:Silk Proteins are biopolymers produced by spinning organisms that have been studied extensively for applications in materials engineering, regenerative medicine, and devices due to their high tensile strength and extensibility. This remarkable combination of mechanical properties arises from their unique semi-crystalline secondary structure and block copolymer features. The secondary structure of Silks is highly sensitive to processing, and can be manipulated to achieve a wide array of material profiles. Studying the secondary structure of Silks is therefore critical to understanding the relationship between structure and function, the strength and stability of Silk-based materials, and the natural fiber synthesis process employed by spinning organisms. However, Silks present unique challenges to structural characterization due to high-molecular-weight protein chains, repetitive sequences, and heterogeneity in intra- and interchain domain sizes. Here, experimental techniques used to study the secondary structure of Silks, the information attainable from these techniques, and the limitations associated with them are reviewed. Ultimately, the appropriate utilization of a suite of techniques discussed here will enable detailed characterization of Silk-based materials, from studying fundamental processing-structure-function relationships to developing commercially useful quality control assessments.
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mass production of biocompatible graphene using Silk nanofibers
ACS Applied Materials & Interfaces, 2018Co-Authors: Xiaoyi Zhang, Ling Wang, David L KaplanAbstract:Mass production of high-quality graphene dispersions under mild conditions impacts the utility of the material for biomedical applications. Various Proteins have been used to prepare graphene dispersions, rare sources, and expensive prices for these Proteins restrict their large-scale utility for the production of graphene. Here, inexpensive Silk Proteins as an abundant resource in nature were used for graphene exfoliation. The Silk Proteins were assembled into hydrophobic nanofibers with negative charge, and then optimized for the production of graphene. Significantly higher concentrations (>8 mg mL–1) and yields (>30%) of graphene dispersions under ambient aqueous conditions were achieved compared with previous protein-assisted exfoliation systems. The exfoliated graphene exhibited excellent stability in water and fetal bovine serum solution, cytocompatibility, and conductivity, suggesting a promising future in biomedical and bioengineering applications.
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Recombinant Silk Production in Bacteria
Reference Module in Materials Science and Materials Engineering, 2016Co-Authors: David L Kaplan, Thomas ScheibelAbstract:Abstract Silks can be defined as secreted Proteins spun into fibrous structures. Characteristically, Silk Proteins are produced in specialized glands and stored in a fluid state in the lumen of the gland. As the fluid passes through the spinning duct, a rapid transformation to the solid state takes place and the Silk becomes water-insoluble. Silk fibers are diverse in function, depending on the biological source, such as spiders or Silkworms. The article describes the recent remarkable progress in understanding Silk genetics, structures and biophysics, as well as the recombinant production of Silk Proteins.
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in vivo bioresponses to Silk Proteins
Biomaterials, 2015Co-Authors: Amy E Thurber, Fiorenzo G Omenetto, David L KaplanAbstract:Silks are appealing materials for numerous biomedical applications involving drug delivery, tissue engineering, or implantable devices, because of their tunable mechanical properties and wide range of physical structures. In addition to the functionalities needed for specific clinical applications, a key factor necessary for clinical success for any implanted material is appropriate interactions with the body in vivo. This review summarizes our current understanding of the in vivo biological responses to Silks, including degradation, the immune and inflammatory response, and tissue remodeling with particular attention to vascularization. While we focus in this review on Silkworm Silk fibroin protein due to the large quantity of in vivo data thanks to its widespread use in medical materials and consumer products, spider Silk information is also included if available. Silk Proteins are degraded in the body on a time course that is dependent on the method of Silk fabrication and can range from hours to years. Silk protein typically induces a mild inflammatory response that decreases within a few weeks of implantation. The response involves recruitment and activation of macrophages and may include activation of a mild foreign body response with the formation of multinuclear giant cells, depending on the material format and location of implantation. The number of immune cells present decreases with time and granulation tissue, if formed, is replaced by endogenous, not fibrous, tissue. Importantly, Silk materials have not been demonstrated to induce mineralization, except when used in calcified tissues. Due to its ability to be degraded, Silk can be remodeled in the body allowing for vascularization and tissue ingrowth with eventual complete replacement by native tissue. The degree of remodeling, tissue ingrowth, or other specific cell behaviors can be modulated with addition of growth or other signaling factors. Silk can also be combined with numerous other materials including Proteins, synthetic polymers, and ceramics to enhance its characteristics for a particular function. Overall, the diverse array of Silk materials shows excellent bioresponses in vivo with low immunogenicity and the ability to be remodeled and replaced by native tissue making it suitable for numerous clinical applications.
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Carbonization of a stable β-sheet-rich Silk protein into a pseudographitic pyroprotein
Nature Communications, 2015Co-Authors: Se Youn Cho, David L Kaplan, Young Soo Yun, Sungho Lee, Dawon Jang, Kyu-young Park, Jae Kyung Kim, Byung Hoon Kim, Kisuk Kang, Hyoung-joon JinAbstract:Silk Proteins are of great interest to the scientific community owing to their unique mechanical properties and interesting biological functionality. In addition, the Silk Proteins are not burned out following heating, rather they are transformed into a carbonaceous solid, pyroprotein; several studies have identified potential carbon precursors for state-of-the-art technologies. However, no mechanism for the carbonization of Proteins has yet been reported. Here we examine the structural and chemical changes of Silk Proteins systematically at temperatures above the onset of thermal degradation. We find that the β-sheet structure is transformed into an sp ^2-hybridized carbon hexagonal structure by simple heating to 350 °C. The pseudographitic crystalline layers grew to form highly ordered graphitic structures following further heating to 2,800 °C. Our results provide a mechanism for the thermal transition of the protein and demonstrate a potential strategy for designing pyroProteins using a clean system with a catalyst-free aqueous wet process for in vivo applications. The strength and stability of Silk Proteins is thought to be related to the high content of β-sheets within their structures. Here, the authors show that when heated at high temperature, and above that of thermal degradation, these β-sheets are transformed into an ordered hexagonal graphitic structure.
Jan Johansson - One of the best experts on this subject based on the ideXlab platform.
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self assembly of spider Silk Proteins is controlled by a ph sensitive relay
Nature, 2010Co-Authors: Glareh Askarieh, Anna Rising, My Hedhammar, Kerstin Nordling, Alejandra Saenz, Cristina Casals, Jan Johansson, Stefan D KnightAbstract:Many Proteins form fibrillar structures at high concentrations, but spider Silk Proteins, with highly repetitive segments flanked by non-repetitive (NR) terminal domains, behave differently. They are remarkably soluble when stored at high concentration yet can convert to extremely sturdy fibres on demand. The molecular mechanism that makes this possible is not yet clear, but two structural studies in this issue provide new clues. Askarieh et al. present the 1.7 A X-ray crystal structure of the N-terminal domain of a dragline spidroin from the nursery web spider Euprosthenops australis. The structure shows how this highly conserved domain can regulate Silk assembly by preventing premature aggregation of spidroins and triggering polymerization as the pH falls along the Silk extrusion duct. Hagn et al. determined the solution structure of the C-terminal NR domain of the dragline Silk protein fibroin 3 from the common orb-weaver Araneus diadematus. They observe a conformational switch, activated by chemical or mechanical stimuli, between storage and assembly forms of the protein. Spider Silk Proteins are remarkably soluble when stored at high concentration and yet can be converted to extremely sturdy fibres, through unknown molecular mechanisms. Here, the X-ray structure of the amino-terminal domain of a Silk protein is presented, revealing how evolutionarily conserved polar surfaces might control self-assembly as the pH is lowered along the spider's Silk extrusion duct. Such a mechanism might be applicable to the design of versatile fibrous materials. Nature’s high-performance polymer, spider Silk, consists of specific Proteins, spidroins, with repetitive segments flanked by conserved non-repetitive domains1,2. Spidroins are stored as a highly concentrated fluid dope. On Silk formation, intermolecular interactions between repeat regions are established that provide strength and elasticity3,4. How spiders manage to avoid premature spidroin aggregation before self-assembly is not yet established. A pH drop to 6.3 along the spider’s spinning apparatus, altered salt composition and shear forces are believed to trigger the conversion to solid Silk, but no molecular details are known. Miniature spidroins consisting of a few repetitive spidroin segments capped by the carboxy-terminal domain form metre-long Silk-like fibres irrespective of pH5. We discovered that incorporation of the amino-terminal domain of major ampullate spidroin 1 from the dragline of the nursery web spider Euprosthenops australis (NT) into mini-spidroins enables immediate, charge-dependent self-assembly at pH values around 6.3, but delays aggregation above pH 7. The X-ray structure of NT, determined to 1.7 A resolution, shows a homodimer of dipolar, antiparallel five-helix bundle subunits that lack homologues. The overall dimeric structure and observed charge distribution of NT is expected to be conserved through spider evolution and in all types of spidroins. Our results indicate a relay-like mechanism through which the N-terminal domain regulates spidroin assembly by inhibiting precocious aggregation during storage, and accelerating and directing self-assembly as the pH is lowered along the spider’s Silk extrusion duct.
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macroscopic fibers self assembled from recombinant miniature spider Silk Proteins
Biomacromolecules, 2007Co-Authors: Margareta Stark, Anna Rising, Stefan Grip, Wilhelm Engstrom, Goran Hjalm, My Hedhammar, Jan JohanssonAbstract:Strength, elasticity, and biocompatibility make spider Silk an attractive resource for the production of artificial biomaterials. Spider Silk Proteins, spidroins, contain hundreds of repeated poly alanine/glycine-rich blocks and are difficult to produce recombinantly in soluble form. Most previous attempts to produce artificial spider Silk fibers have included solubilization steps in nonphysiological solvents. It is here demonstrated that a miniature spidroin from a protein in dragline Silk of Euprosthenops australis can be produced in a soluble form in Escherichia coli when fused to a highly soluble protein partner. Although this miniature spidroin contains only four poly alanine/glycine-rich blocks followed by a C-terminal non-repetitive domain, meter-long fibers are spontaneously formed after proteolytic release of the fusion partner. The structure of the fibers is similar to that of dragline Silks, and although self-assembled from recombinant Proteins they are as strong as fibers spun from redissolved...
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macroscopic fibers self assembled from recombinant miniature spider Silk Proteins
Biomacromolecules, 2007Co-Authors: Margareta Stark, Anna Rising, Stefan Grip, Wilhelm Engstrom, Goran Hjalm, My Hedhammar, Jan JohanssonAbstract:Strength, elasticity, and biocompatibility make spider Silk an attractive resource for the production of artificial biomaterials. Spider Silk Proteins, spidroins, contain hundreds of repeated poly alanine/glycine-rich blocks and are difficult to produce recombinantly in soluble form. Most previous attempts to produce artificial spider Silk fibers have included solubilization steps in nonphysiological solvents. It is here demonstrated that a miniature spidroin from a protein in dragline Silk of Euprosthenops australis can be produced in a soluble form in Escherichia coli when fused to a highly soluble protein partner. Although this miniature spidroin contains only four poly alanine/glycine-rich blocks followed by a C-terminal non-repetitive domain, meter-long fibers are spontaneously formed after proteolytic release of the fusion partner. The structure of the fibers is similar to that of dragline Silks, and although self-assembled from recombinant Proteins they are as strong as fibers spun from redissolved Silk. Moreover, the fibers appear to be biocompatible because human tissue culture cells can grow on and attach to the fibers. These findings enable controlled production of high-performance biofibers at large scale under physiological conditions.
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n terminal nonrepetitive domain common to dragline flagelliform and cylindriform spider Silk Proteins
Biomacromolecules, 2006Co-Authors: Anna Rising, Wilhelm Engstrom, Goran Hjalm, Jan JohanssonAbstract:Spider Silk has been extensively studied for its outstanding mechanical properties. Partial intermediate and C-terminal sequences of different spider Silk Proteins have been determined, and during the past decade also N-terminal domains have been characterized. However, only some of these N-terminal domains have been reported to contain signal peptides, leaving the mechanism whereby they enter the secretory pathway open to speculation. Here we present the sequence of a 394-residue N-terminal region of the Euprosthenops australis major ampullate spidroin 1 (MaSp1). A close comparison with published sequences from other species revealed the presence of N-terminal signal peptides followed by an approximately 130-residue nonrepetitive domain. From secondary structure predictions, helical wheel analysis, and circular dichroism spectroscopy this domain is concluded to contain five α-helices and is a conserved constituent of hitherto analyzed dragline, flagelliform, and cylindriform spider Silk Proteins.
Randolph V Lewis - One of the best experts on this subject based on the ideXlab platform.
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Method for the Destruction of Endotoxin in Synthetic Spider Silk Proteins.
Scientific Reports, 2018Co-Authors: Richard E. Decker, Thomas I. Harris, Dylan R. Memmott, Randolph V Lewis, Chris Peterson, Justin A. JonesAbstract:Although synthetic spider Silk has impressive potential as a biomaterial, endotoxin contamination of the spider Silk Proteins is a concern, regardless of the production method. The purpose of this research was to establish a standardized method to either remove or destroy the endotoxins present in synthetic spider Silk Proteins, such that the endotoxin level was consistently equal to or less than 0.25 EU/mL, the FDA limit for similar implant materials. Although dry heat is generally the preferred method for endotoxin destruction, heating the Silk Proteins to the necessary temperatures led to compromised mechanical properties in the resultant materials. In light of this, other endotoxin destruction methods were investigated, including caustic rinses and autoclaving. It was found that autoclaving synthetic spider Silk protein dopes three times in a row consistently decreased the endotoxin level 10–20 fold, achieving levels at or below the desired level of 0.25 EU/mL. Products made from triple autoclaved Silk dopes maintained mechanical properties comparable to products from untreated dopes while still maintaining low endotoxin levels. Triple autoclaving is an effective and scalable method for preparing synthetic spider Silk Proteins with endotoxin levels sufficiently low for use as biomaterials without compromising the mechanical properties of the materials.
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Silkworms transformed with chimeric Silkworm spider Silk genes spin composite Silk fibers with improved mechanical properties
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Florence Teule, Yungen Miao, Malcolm J Fraser, Bonghee Sohn, Joe J Hull, Randolph V Lewis, Donald L JarvisAbstract:The development of a spider Silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider Silk Proteins into fibers. Thus, we used piggyBac vectors to create transgenic Silkworms encoding chimeric Silkworm/spider Silk Proteins. The Silk fibers produced by these animals were composite materials that included chimeric Silkworm/spider Silk Proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental Silkworm Silk fibers and as tough as native dragline spider Silk fibers. These results demonstrate that Silkworms can be engineered to manufacture composite Silk fibers containing stably integrated spider Silk protein sequences, which significantly improve the overall mechanical properties of the parental Silkworm Silk fibers.
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analysis of the conserved n terminal domains in major ampullate spider Silk Proteins
Biomacromolecules, 2005Co-Authors: Dagmara Motriuksmith, Alyson J Smith, Cheryl Y Hayashi, Randolph V LewisAbstract:Major ampullate Silk, also known as dragline Silk, is one of the strongest biomaterials known. This Silk is composed of two Proteins, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2). Only partial cDNA sequences have been obtained for these Proteins, and these sequences are toward the C-terminus. Thus, the N-terminal domains have never been characterized for either protein. Here we report the sequence of the N-terminal region of major ampullate Silk Proteins from three spider species: Argiope trifasciata, Latrodectus geometricus, and Nephila inaurata madagascariensis. The amino acid sequences are inferred from genomic DNA clones. Northern blotting experiments suggest that the predicted 5' end of the transcripts are present in fibroin mRNA. The presence of more than one Met codon in the N-terminal region indicates the possibility of translation of both a long and a short isoform. The size of the short isoform is consistent with the published, cDNA based, N-terminal sequence found in flagelliform Silk. Analyses comparing the level of identity of all known spider Silk N-termini show that the N-terminus is the most conserved part of Silk Proteins. Two DNA sequence motifs identified upstream of the putative transcription start site are potential Silk fibroin promoter elements.
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Elastic Spider Silk Proteins.
1998Co-Authors: Randolph V LewisAbstract:Abstract : Flagelliform Silk Proteins have been studied by cloning the cDNAs for the major protein in that Silk as well as the gene. The protein consists of three sequence segments which compose a repeat These repeats appear numerous times in the protein The three segments are: 1) GPGGX; 2) GGX; and 3) a highly conserved non-Silk-like "spacer" sequence. The gene is composed of the same repeats combined with a highly conserved intron. Two major publications have been published and four patents awarded during the tenure of this grant.
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Spider Silk Proteins
MRS Proceedings, 1992Co-Authors: Mike Hinman, Zhengyu Dong, Ming Xu, Randolph V LewisAbstract:Abstract : Spider Silk Proteins from major and minor ampullate Silk have been sequenced via their cDNAs. One of the Proteins from major ampullate Silk has been expressed in bacteria to levels of 10-20 mg/mL. Studies using fiber X-ray diffraction and solid state NMR have been used to study the structure of the Proteins in the fiber. Spider, Silk, Proteins, Expression, X-ray diffraction NMR.
Kisuk Kang - One of the best experts on this subject based on the ideXlab platform.
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Carbonization of a stable β-sheet-rich Silk protein into a pseudographitic pyroprotein
Nature Communications, 2015Co-Authors: Se Youn Cho, David L Kaplan, Young Soo Yun, Sungho Lee, Dawon Jang, Kyu-young Park, Jae Kyung Kim, Byung Hoon Kim, Kisuk Kang, Hyoung-joon JinAbstract:Silk Proteins are of great interest to the scientific community owing to their unique mechanical properties and interesting biological functionality. In addition, the Silk Proteins are not burned out following heating, rather they are transformed into a carbonaceous solid, pyroprotein; several studies have identified potential carbon precursors for state-of-the-art technologies. However, no mechanism for the carbonization of Proteins has yet been reported. Here we examine the structural and chemical changes of Silk Proteins systematically at temperatures above the onset of thermal degradation. We find that the β-sheet structure is transformed into an sp ^2-hybridized carbon hexagonal structure by simple heating to 350 °C. The pseudographitic crystalline layers grew to form highly ordered graphitic structures following further heating to 2,800 °C. Our results provide a mechanism for the thermal transition of the protein and demonstrate a potential strategy for designing pyroProteins using a clean system with a catalyst-free aqueous wet process for in vivo applications. The strength and stability of Silk Proteins is thought to be related to the high content of β-sheets within their structures. Here, the authors show that when heated at high temperature, and above that of thermal degradation, these β-sheets are transformed into an ordered hexagonal graphitic structure.
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carbonization of a stable β sheet rich Silk protein into a pseudographitic pyroprotein
Nature Communications, 2015Co-Authors: Se Youn Cho, Young Soo Yun, Sungho Lee, Dawon Jang, Kyu-young Park, Jae Kyung Kim, Byung Hoon Kim, Kisuk KangAbstract:Silk Proteins are of great interest to the scientific community owing to their unique mechanical properties and interesting biological functionality. In addition, the Silk Proteins are not burned out following heating, rather they are transformed into a carbonaceous solid, pyroprotein; several studies have identified potential carbon precursors for state-of-the-art technologies. However, no mechanism for the carbonization of Proteins has yet been reported. Here we examine the structural and chemical changes of Silk Proteins systematically at temperatures above the onset of thermal degradation. We find that the β-sheet structure is transformed into an sp(2)-hybridized carbon hexagonal structure by simple heating to 350 °C. The pseudographitic crystalline layers grew to form highly ordered graphitic structures following further heating to 2,800 °C. Our results provide a mechanism for the thermal transition of the protein and demonstrate a potential strategy for designing pyroProteins using a clean system with a catalyst-free aqueous wet process for in vivo applications.
