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Animal-derived xenogeneic biomaterials utilized in different surgeries are promising for various applications in tissue engineering. However, tissue decellularization is necessary to attain a bioactive extracellular matrix (ECM) that can be safely transplanted. The main objective of the present study is to assess the structural integrity, biocompatibility, and potential use of various acellular biomaterials for tissue engineering applications. Hence, a bovine pericardium (BP), porcine pericardium (PP), and porcine tunica vaginalis (PTV) were decellularized using a Trypsin, Triton X (TX), and sodium dodecyl sulfate (SDS) (Trypsin + TX + SDS) protocol. The results reveal effective elimination of the cellular antigens with preservation of the ECM integrity confirmed via staining and electron microscopy. The elasticity of the decellularized PP (DPP) was markedly (p < 0.0001) increased. The tensile strength of DBP, and DPP was not affected after decellularization. All decellularized tissues were biocompatible with persistent growth of the adipose stem cells over 30 days. The staining confirmed cell adherence either to the peripheries of the materials or within their matrices. Moreover, the in vivo investigation confirmed the biocompatibility and degradability of the decellularized scaffolds. Conclusively, Trypsin + TX + SDS is a successful new protocol for tissue decellularization. Moreover, decellularized pericardia and tunica vaginalis are promising scaffolds for the engineering of different tissues with higher potential for the use of DPP in cardiovascular applications and DBP and DPTV in the reconstruction of higher-stress-bearing abdominal walls.

Bioabsorbable arterial grafts can potentially improve patency and neovessel formation; however, their application in clinical settings has not been realized. In this study, we developed bioabsorbable gradient sheets based on silk fibroin (SF) and polyvinyl alcohol (PVA) with a core-shell nanofibrous structure. This gradient sheet was expected to promote vascular remodeling while we maintained its physical properties and a gradual degrading process from the luminal surface. ESP was conducted at various flow rates for SF and PVA to achieve the multilayer gradient structure. Furthermore, the elasticity of the gradient sheet could be increased by increasing the PVA flow rate; however, this reduced the tensile strength of the core-shell fibers. Notably, the physical properties of the gradient sheet did not degrade even after 7 days of immersion in a phosphate buffer saline solution, which indicates that the structure could maintain its structural integrity while resisting arterial pressure. In vitro experiments revealed that the number of endothelial cells attached to the SF/PVA sheet was notably higher than that on the cell-culture dish. The gradient sheets were implanted in rat abdominal aortas and explanted after 14 days to confirm acute-phase patency and vascular remodeling. The gradient sheets constructed with SF composed of polyurethane and PVA improved the ease of handling of the material, and these sheets resulted in a favorable vascular remodeling outcome. Our results strongly suggest that the SF/PVA-based gradient sheets described in this study can serve as a novel design for bioabsorbable arterial grafts upon further modifications.

We propose a method to determine the limiting value of 1H spin–spin relaxation time (T2) for assessing the lifetime of thermally aged acrylonitrile butadiene rubber (NBR) parts. So far, it has been difficult to determine the limiting values of T2 without performing other experiments such as mechanical property measurements and air tightness tests. We exposed two types of NBR diaphragm sheets to air at 100 °C for up to 7540 h and 6000 h, respectively. After that, we tested their air-tightness and we measured T2 of the virgin and aged diaphragms. To evaluate the temperature dependence of T2, T2 measurements were performed at 20, 40, and 60 °C. Additionally, we observed the 13C Nuclear Magnetic Resonance (NMR) spectra, toluene swelling ratio, acetone soluble fraction, and glass transition temperature for only one type of the diaphragm sheets. The diaphragm aged for 7540 h had lost sealability, and all the results indicated that this was caused by the degradation of NBR. The 13C NMR spectra clearly revealed that the degradation of NBR proceeded in three stages: self-cross-linking reactions in the initial stage, concurrent reactions of cross-linking and molecular chain scission in the second stage, and an increased number of reactions of cross-linking and molecular chain scission in the final stage. The results of the T2 measurements showed that T2 is useful not only to evaluate the complicated degradation behavior quantitatively, but also to determine the different limiting values of T2 itself corresponding to large and small safety margins. Finally, we demonstrated that the temperature dependence of T2 is an effective parameter for quantitatively predicting the ultimate lifetime at each aging temperature with no safety margin for various thermally aged NBR rubber sheets.

Purpose To understand the complication and histopathological characteristics between the Silk Fibroin/Polyurethanes (SF/PU) and the host response, and to unveil the compatibility of the patch in diabetes individuals. Methods Rats were divided into DM and control (CT) groups, and the DM group was induced with streptozotocin. All groups underwent the SF/PU patch implantation in the abdominal aorta, and the implanted patches were evaluated at one, two, three, and four weeks after implantation. Results DM group had more fibrosis formation and a delayed endothelialization compared to the CT group. There was no evidence of chronic inflammation in both DM and CT groups. Conclusions Fibrosis in hyperglycemic individuals could promote the formation of new vascular structures in the implanted patch such as endothelial and vascular smooth muscle cells. In summary, the SF/PU patch was no serious complications when implanted under hyperglycemia, and the patch was suitable to implant in diabetes mellitus.

Films from silk fibroin protein are one of the most promising biomaterials because of their exquisite balance between mechanical properties and biocompatibility. Numerous schemes have been proposed for processing fibroin film, utilizing liquid silk fibroin (LSF) or regenerated silk fibroin (RSF). The films cast from LSF or RSF in the solution state are water-soluble, and therefore require postproduction treatment inducing β-sheet formation, to render them insoluble in water. Many kinds of postproduction treatments, using alcohol-water solution, water vapor, or controlled temperature, have been developed. However, the tuning and reproducibility of such treatments are quite sensitive and frequently render the fibroin films less flexible or even brittle because of the formation of an over content of β-sheet. To overcome this, we developed a novel scheme for fibroin processing using silk-gland fibroin (SGF). The essence of this scheme is to create a softly solidified fibroin-gel state of the silk glands with an imperfect β-sheet structure, by treating them with an ethanol/water mixture. Such a fibroin gel was found to dissolve in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The SGF film cast from the HFIP solution shows a flexible and water-insoluble nature with high reproducibility. In addition to this improvement, the SGF film produced by this method contains a significantly low level of residual HFIP molecules compared to the traditional RSF films prepared from an HFIP solution. The mechanism underlying these advantageous characteristics was investigated from the structural viewpoint, by using techniques such as

Medical sheets are useful in surgically repair vascular disease. To avoid long-term side effects, they are to be replaced with regenerated tissue after implantation. Silk fibroin is a fibrous protein secreted by silkworm. The advantage of silk fibroin is its biocompatibility and has been used as regenerative artificial materials. The problem of its biodegradability is that the effect is time consuming. In this study, SVVYGLR peptide was used to expect promoting cell migration and accelerating the biodegradation of silk fibroin. Silk fibroin and polyurethane-based medical sheets with or without SVVYGLR peptide were implanted in rat abdominal aorta (silk fibroin/polyurethane/SVVYGLR peptide versus silk fibroin/polyurethane). The result of histological evaluation indicated that the new cell layer created under both sheets was composed of endothelial cells, smooth muscle, and fibroin in both sheets and similar to a native vessel. Both sheets did not show any excessive inflammation or calcification, and moderate biodegradability was observed. The decrease of silk fibroin indicated the biodegradability of all sheets. Silk fibroin/polyurethane/SVVYGLR peptide had many small vessels in the regenerated tissue than silk fibroin/polyurethane. This appearance indicated that SVVYGLR peptide promoted the angiogenesis in the regenerative tissue. This study suggested that SVVYGLR peptide could give the angiogenic-promoting activity to silk fibroin-based vascular repairing sheet.

Interest in vascular grafts has recently grown because more patients are undergoing procedures that involve these grafts. However, smaller grafts with diameters

Films from silk fibroin protein are one of the most promising biomaterials because of their exquisite balance between mechanical properties and biocompatibility. Numerous schemes have been proposed for processing fibroin film, utilizing liquid silk fibroin (LSF) or regenerated silk fibroin (RSF). The films cast from LSF or RSF in the solution state are water-soluble, and therefore require postproduction treatment inducing β-sheet formation, to render them insoluble in water. Many kinds of postproduction treatments, using alcohol–water solution, water vapor, or controlled temperature, have been developed. However, the tuning and reproducibility of such treatments are quite sensitive and frequently render the fibroin films less flexible or even brittle because of the formation of an over content of β-sheet. To overcome this, we developed a novel scheme for fibroin processing using silk-gland fibroin (SGF). The essence of this scheme is to create a softly solidified fibroin-gel state of the silk glands with ...

Silk fibroin (SF) possesses several characteristics that are favorable for tissue engineering. However, the mechanical properties must be modified in some cases. For instance, soft tissues such as those of the circulatory system are more elastic than tissue-engineered materials. The development of polymer blends is a simple method with the potential to provide materials with extended useful properties. In this study, we developed a non-woven SF and thermoplastic polyurethane-blend sheet from a polymer solution. The structure and miscibility of the blend sheet was evaluated using solid-state nuclear magnetic resonance (NMR) methods. In the 13C cross-polarization and magic angle spinning NMR spectra, no peak shift was observed between the pure and blended samples. The miscibility of the sample was investigated using proton spin-lattice relaxation times in the laboratory frame (T1H). The T1H measurement clearly revealed that the molecular chains of SF and Pellethane exist in close proximity of several tens of nanometers. The development of polymer blends is a simple method with the potential to provide materials with extended useful properties. Here, the SF and Pellethane were observed to be highly miscible with each other even though SF maintained the strong β-sheet crystal structure. This result suggests that SF/Pellethane-blended sheets possess strength that is derived from SF and elasticity that is derived from Pellethane. Therefore, this blended sheet should be a good medical material for soft tissue engineering.

Samia cynthia ricini (S. c. ricini) is one of the wild silkworms. Their silk fibroins have been paid attention as potentially valuable biomedical materials as well as Bombyx mori silk fibroins, but detailed information on the packing arrangement of the fibers is still not currently well understood at a molecular level. In this study, 34 mer model peptides, GGAGGGYGGDGG(A)12GGAGDGYGAG with different 13C labeled positions have been synthesized as a typical sequence of the primary structure of S. c. ricini silk fibroins made up of tandemly repeated sequences of polyalanine as the crystalline region and glycin-rich sequences as the non-crystalline region. The heterogeneous structure was obtained from the determination of the fraction of several conformations depending on the position of the Ala residue by 13C cross polarization/magic angle spinning NMR. The packing arrangement was studied by 13C dipolar assisted rotational resonance NMR and packing in a staggered arrangement rather than a rectangular arrangement of this peptide with an anti-parallel β-sheet structure was clarified, which is in good agreement with our previous report on the packing arrangement of (Ala)7 with an anti-parallel β-sheet structure.

Formation of the &alpha, helical conformation in the poly-l-alanine (PA) sequence regions, subsequent structural transition to &beta, sheet during natural spinning, and presence of residual &alpha, helices in Samia cynthia ricini (S. c. ricini) native silk fiber have been experimentally proven. However, the aggregation state of the residual &alpha, helices, and their influence on the mechanical deformation behavior in native fiber remain unclear. Here we show that the &alpha, helices form an ordered aggregation state with a hexagonal packing in the aqueous solution, some of which remain during natural spinning. X-ray scattering and differential scanning calorimetry (DSC) analyses revealed occurrence of a structural transition of the residual &alpha, helices to the &beta, sheet structure, accompanied by disappearance of the plateau region in the force-strain curve, due to heat-treatment at ~220 &deg, C. On the basis of X-ray scattering before and after tensile stretching of S. c. ricini native silk, a direct connection between the plateau region and the &alpha, helix to &beta, sheet structural transition was confirmed. Our findings demonstrate the importance of the PA sequence regions in fiber structure formation and their influence on the tensile deformation behavior of S. c. ricini silk, features believed to be essentially similar in other saturniid silks. We strongly believe the residual ordered &alpha, helices to be strategically and systematically designed by S. c. ricini silkworms to impart flexibility in native silk fiber. We anticipate that these knowledge forms a basis for fruitful strategies in the design and development of amino acid sequences for artificial silks with desired mechanical properties.

This paper presents the applicability of proton spin–spin relaxation time, T 2 , for the evaluation of the degradation of sealability for rubber seals. So far, compression set (CS) has been utilized to evaluate the degree of degradation of rubber seals. However, the reliability of CS for thin rubber seals is relatively low. Therefore, in this paper, we investigated the applicability of T 2 as a parameter for the quantitative evaluation of the degradation of sealability instead of CS. The correlations between T 2 and cross-link density, plasticizer concentration and CS for two acrylonitrile butadiene rubber (NBR) samples were discussed. The samples have different compounds and shapes: plate and O-ring. We exposed them to air and nitrogen atmospheres at high temperatures of 60, 80 and 100 °C for up to 33,000 h. The short spin–spin relaxation time, T 2 S , was shortened with aging and correlated well with the cross-link density, plasticizer concentration and CS, regardless of the atmosphere. These results revealed that T 2 S is a useful parameter for the quantitative evaluation of the degradation of sealability.

We have demonstrated that solid-state and time-domain Nuclear Magnetic Resonance spectroscopy (NMR) techniques are effective in monitoring the degradation site of poly (urea-urethane). We prepared two types of commercial poly (urea-urethane), Urethane-A and Urethane-B, and aged them at 100 °C for up to 10000 h. We performed tensile strength tests and solid-state NMR experiments, in which 13C and 15N NMR spectra and T2 were collected. The tensile strength of Urethane-A decreased, increased and decreased again, whereas that of Urethane-B decreased at a lower rate with aging. Solid-state NMR experiments clearly showed that the degradation of Urethane-A was caused by molecular chain scission and cross-linking in its urea bonds, and that of Urethane-B occurred owing to molecular chain scission in its urethane bonds. T2 had three components, short T2 (T2S), intermediate T2 (T2I) and long T2 (T2L), whose fractions were xS, xI and xL, respectively. We have demonstrated that molecular chain scission in urea bonds decreases xS and increases xL, and that in urethane bonds increases T2I and T2L without changing all the T2 fractions.

Patch grafts are widely used in various kind of vascular surgeries such as detect repair or dilation of vascular stenosis. Expanded polytetrafluoroethylene (ePTFE) patches are flexible and handle well, but have shown problems with calcification as they are non-bioabsorbable and therefore permanently remain in the body. It is important to develop an alternative biocompatible patch. Silk fibroin (SF) was developed as a biocompatible material, but it lacks of the elasticity required for surgery as a patch. Polyurethane (PU) is also a well-known elastomer so this study focused on the SF and the PU blend materials with a weight ratio of 5:5 (SF/PU). To evaluate the SF/PU patch, the patches were implanted into the abdominal aortas of rats, using the ePTFE patch in the control group. Because it was more flexible the SF/PU patch was easier to implant than the ePTFE patch. At 1 week after implantation, the SF/PU patch had been infiltrated with cells and collagen fiber. The ePTFE control patch did not accumulate collagen fiber until 3 months and calcification occurred at 4 weeks. The SF/PU patch did not present any signs of calcification for 3 months. This study addressed the problems associated with using SF in isolation and showed that the SF/PU patch can be considered as a useful alternative to the ePTFE to overcome the problem of calcification.

The surgical sheets that are currently used for congenital cardiovascular surgery have several drawbacks, including material deterioration, calcification, and pseudo-intimal proliferation resulting in hemodynamic disturbance. The aim of this study was to evaluate a newly developed sheet made from a combination of silk fibroin (SF) and a synthetic polymer, thermoplastic polyurethane (TPU), for surgical use. The hybrid SF/TPU sheet was a non-woven fabric with nanofibers that was made using the electrospinning method. The mechanical properties of the SF/TPU sheet were characterized. To determine its biocompatibility, part of the wall of the canine descending aorta was replaced with a SF/TPU sheet as a patch. The patches were removed after 3 months and a histological examination was performed. The flexibility, water permeability, and suture retention strength of the SF/TPU sheet were excellent and equivalent to those of existing sheets. The SF/TPU sheet had excellent handling properties and fit well into the vascular wall without needle hole bleeding. The histological examination revealed that the intimal tissue was restored well over the intraluminal surface of the explanted SF/TPU sheet, the absence of calcium deposition, and minimal inflammatory reaction, without signs of degradation. The SF/TPU sheet had excellent mechanical properties and tissue biocompatibility. These favorable features and possible biodegradability of the SF portion warrant a long-term follow-up study.

Samia cynthia ricini (S. c. ricini) is one of the wild silkworms. Their silk fibroins have been paid attention as potentially valuable biomedical materials as well as Bombyx mori silk fibroins, but detailed information on the packing arrangement of the fibers is still not currently well understood at a molecular level. In this study, 34 mer model peptides, GGAGGGYGGDGG(A)

Silk is a protein fiber used to weave fabrics and as a biomaterial in medical applications. Recently, genetically modified silks have been produced from transgenic silkworms. In the present study, transgenic silkworms for the mass production of three colors of fluorescent silks, (green, red, and orange) are generated using a vector originating from the fibroin H chain gene and a classical breeding method. The suitability of the recombinant silks for making fabrics is investigated by harvesting large amounts of the cocoons, obtained from rearing over 20 thousand silkworms. The application of low temperature and a weakly alkaline solution for cooking and reeling enables the production of silk fiber without loss of color. The maximum strain tolerated and Young's modulus of the fluorescent silks are similar to those of ordinary silk, although the maximum stress value of the recombinant silk is slightly lower than that of the control. Fabrics with fluorescent color are demonstrated using the recombinant silk, with the color persisting for over two years. The results indicate that large amounts of genetically modified silk can be made by transgenic silkworms, and the silk is applicable as functional silk fiber for making fabrics and for use in medical applications.

Amyloid A (AA) amyloidosis can be induced by the administration of amyloid fibrils to animals under inflammatory conditions. Silk fibroin (SF) is a main component protein of bombic silk and has amyloid-like features. The amyloidogenesis of SF solution in mice has been previously reported. Recently, the biochemical properties of silk have attracted increasing attention, and research and development have been undertaken regarding applications other than as a clothing material. However, the risk of AA amyloidosis from exposure to SF-related products is unknown. In this study, we examined the amyloidogenesis of several SF-related products that vary in preparation method or route of injection in a mouse model of amyloidosis. The results revealed that amyloid deposits were rarely observed in mice exposed to SF solution or feed supplemented with SF powder. On the other hand, heavy amyloid deposits were observed in some mice implanted with SF non-woven fabric by abdominal operation. Congo red staining of SF solutions under polarized light and electron microscopy indicated that SF solution in this study had no amyloid-like structures. We found that SF-related products occasionally promote amyloidogenesis, but have a low potential for amyloidosis.

The local structure of the acidic-treated model peptides,(E)8GGLGGQGAG(A)6GGAGQGGYGG, derived from the consensus sequence of Nephila clavipes fibroin major ampullate spidroin 1 was determined using two-dimensional proton-driven spin-diffusion solid-state NMR under off-magic-angle spinning coupled with 13C isotope double labeling of specific residues. We observed a positional dependence on the torsion angles of Ala residues in the β sheet. Torsion angle of the Ala residue at the center of poly-Ala domain concentrates around at (ϕ, ψ) = (−150°, 150°), while that of its periphery is distorted and more distributed.

In this paper, we performed solid-state nuclear magnetic resonance (NMR) measurements, including T1H and T1ρ, of silk fibroin (SF)/polyurethane (PU) composites to examine their possible use as a material for artificial vascular grafts. In the development of new artificial vascular grafts made from SF/PU, it is important to examine the miscibility of the composites and their molecular dynamics, because these properties are intimately involved in the resulting physical properties of the resulting vascular graft. The T1H measurements showed that the domain size of the SF/PU=1:1 composite is smaller than the domain size of the 1:10 and 1:2 composites, indicating that the molecular miscibility between SF and PU are partially in close proximity, particularly in the SF/PU=1:1 composite. Additionally, we observed that the molecular motion of the soft segment of PU in the SF/PU composites becomes slow, suggesting that the soft segment of PU interacts with SF to some extent. These analyses provided basic structural information for the development of silk-based artificial vascular grafts using PU. In this study, we investigated the molecular structure of the composite material of silk fibroin and polyurethane as coating agent of vascular grafts by using solid-state nuclear magnetic resonance (NMR) techniques. To elucidate the structure and dynamics, 13C cross-polarization (CP)/magic angle spinning (MAS) NMR studies including NMR relaxation experiments were performed. These observations showed that there is a partial interacted portion between soft segment of PU and SF, in particular, the domain size of PU:SU=1:1 composite was smaller than that of the other composites. This study gave basic information on development of new silk-like materials for vascular grafts.

Bombyx mori silk fibroin with the main sequence (Ala-Gly-Ser-Gly-Ala-Gly)n is a promising scaffold for bone regeneration not only on account of its excellent mechanical property as a structural matrix, but also for its slow biodegradability with adequate control of hydroxyapatite (HAP) deposition. Seeking to develop a material that might stimulate bone regeneration, we prepared a recombinant calcium binding-amphipathic silk-like protein [(Glu)8(Ala-Gly-Ser-Gly-Ala-Gly)4]4 by expression in E. coli. We also prepared the homologous peptide (Glu)8(Ala-Gly-Ser-Gly-Ala-Gly)4 by solid phase synthesis. The poly-L-glutamic acid was introduced into both protein and peptide because this sequence is involved in HAP-nucleating domains of bone sialoprotein. The recombinant protein was shown to bind relatively large quantities of Ca2+ ions in solution by a spectrophotometric assay and in the solid state by X-ray photoelectron spectroscopy. Changes in the electronic structure and local conformation of the peptide resulting from Ca2+ binding were studied using 13C solution NMR, especially 13C chemical shifts. We obtained evidence that Ca2+ bound to the poly-L-glutamic acid domains but not to the predominantly hydrophobic (Ala-Gly-Ser-Gly-Ala-Gly)4 domains. A remarkable conformational change induced by adsorption of the synthetic peptide on the HAP surface was also demonstrated using 13C solid state NMR.

Silk fibroin fiber has a long history of use in sutures because of its high strength and toughness. In the work reported in this paper, small-diameter vascular grafts 1.5 mm in diameter and 10 mm in length were prepared by coating a double-raschel knitted silk fiber graft with silk fibroin aqueous solution containing poly(ethylene glycol diglycidyl ether) as a cross-linking agent. The most important character of silk fibroin graft is remodeling, which is never observed for polyester fiber or expanded polytetrafluoroethylene grafts. The double-raschel knitted silk fiber graft with coating has sufficient physical strength and protects the ladder from the end in the implantation process. The coating also gives protection against leakage of blood from the graft, and elasticity to the graft. Eight weeks after implantation of the grafts in rat abdominal aorta, early formation of thrombosis was avoided.

In our previous study, we have proposed a lamellar structure for Ala-Gly repeated copolymeric peptide, a model for crystalline region of Bombyx mori silk fibroin. Here, we propose the structure of (AGSGAG)5 with silk II form which is a more mimic of the crystalline region of B. mori silk fibroin than (AG)15. The local structure for each Ala residue was determined from 13C CP/MAS NMR spectra of 10 different [3-13C]Ala-(AGSGAG)5 peptides differing in their 13C labeling positions. The highest field peak for the Ala Cβ carbon (16.7 ppm) assigned to a distorted β-turn structure and/or random coil changes significantly depending on the 13C labeling position. In addition, the fractions of the random coil and/or distorted β-turn component of each Ser residue were determined by REDOR experiments from the 13C−15N atomic distances of five versions of the above peptide with different [1-13C]Gly-Ser-[15N]Gly positions. By combining the structural information of Ala and Ser residues from solid state NMR, with statistic...

Peptides with a combination of hydrophilic and hydrophobic sequences mimicking the primary structure of Bombyx mori silk fibroin were synthesized and studied in the solid state by NMR using 13C selective labeling coupled with 13C conformation-dependent chemical shifts and 2D solid-state spin-diffusion NMR. The hydrophilic sequence was poly(l-glutamic acid) (E)n, and the hydrophobic one was the consensus sequence of the crystalline fraction of B. mori silk fibroin, (AGSGAG)4. The balance of hydrophilic and hydrophobic characters of the peptide was controlled by changing the relative length, n, of (E)n from 4 to 8. When n = 4 and 5, the structure of the hydrophobic sequence is basically Silk I (the structure of B. mori silk fibroin before spinning in the solid state), and the polyglutamate sequences are random coil. On the other hand, when n = 6−8, the structure of the polyglutamate sequence changes gradually from random coil to β-sheet, and the hydrophobic sequence adopts a mixture of β-sheet and random co...

Amyloid beta-peptide (Abeta) is a major component of plaques in Alzheimer's disease, and formation of senile plaques has been suggested to originate from regions of neuronal membrane rich in gangliosides. We analyzed the mode of interaction of Abeta with lipid bilayers by multinuclear NMR using (31)P nuclei. We found that Abeta (1-40) strongly perturbed the bilayer structure of dimyristoylphosphatidylcholine (DMPC), to form a non-lamellar phase (most likely micellar). The ganglioside GM1 potentiated the effect of Abeta (1-40), as viewed from (31)P NMR. The difference of the isotropic peak intensity between DMPC/Abeta and DMPC/GM1/Abeta suggests a specific interaction between Abeta and GM1. We show that in the DMPC/GM1/Abeta system there are three lipid phases, namely a lamellar phase, a hexagonal phase and non-oriented lipids. The latter two phases are induced by the presence of the Abeta peptide, and facilitated by GM1.

In an attempt to produce mineralized composite materials with potential use as biomaterials or scaffolds for tissue engineering, we designed silklike peptides based on Ala-Gly repeated sequences with a lamellar structure and Asp as a Ca binding site in the turn part as in Tirrell's work (for example: Macromolecules 1996, 29, 1540−1553). We further modified the design of the lamella structure by introducing a Ser residue between (GlyAla)3 and (AlaGly)3 sequences. At first, we synthesized three labeled versions of 41SDSDS, (GlyAla)3Ser(AlaGly)3Asp(GlyAla)3Ser(AlaGly)3Asp(GlyAla)3Ser(AlaGly)3, with 13C labeling in different positions to characterize the lamellar structure using 13C CP/MAS and spin-diffusion solid-state NMR. The β-sheet fraction in Ala residues increased with increased distance from the Asp residue in the turn part. The introduced Ser residue took almost 100% β-sheet structure probably because it forms an extra hydrogen bond stabilizing the stem part of (AlaGly)n. Thus, position-selective an...

Lamellar structure of poly(Ala-Gly) or (AG) n in the solid was examined using 13 C solid-state NMR and statistical mechanical approaches. Two doubly labeled versions, [1- 13 C]Gly 14 [1- 13 C]Ala 15 - and [1- 13 C]-Gly 18 [1- 13 C]Ala 19 of (AG) 15 were examined by two-dimensional (2D) 13 C spin diffusion NMR in the solid state. In addition five doubly labeled [ 15 N, 13 C]-versions of the same peptide, (AG) 15 and 15 versions labeled [3- 13 C] in each of the successive Ala residues were utilized for REDOR and 13 C CP/MAS NMR measurements, respectively. The observed spin diffusion NMR spectra were consistent with a structure containing a combination of distorted β-turns with a large distribution of the torsion angles and antiparallel β-sheets. The relative proportion of the distorted /3-turn form was evaluated by examination of 13 C CP/ MAS NMR spectra of [3- 13 C]Ala-(AG) 15 . In addition, REDOR determinations showed five kinds of atomic distances between doubly labeled 13 C and 15 N nuclei which were also interpreted in terms of a combination of (3-sheets and β-turns. Our statistical mechanical analysis is in excellent agreement with our Ala Cβ 13 C CP/MAS NMR data strongly suggesting that (AG)15 has a lamellar structure.

The calcium-binding sites of calbindin D(9k) have a helix-loop-helix motif. In this study, the helix motifs were replaced by several Ala-Gly repeating regions designed on the basis of the primary sequences of several silk fibroins. The synthesized peptides were treated with several organic solvents to modify the secondary structure of the Ala-Gly repeating regions. The local structures of the Ala-Gly repeating regions, as well as the calcium-binding motif, D(9k)-loop (D(9k)L), were determined by (13)C CP/MAS NMR spectroscopy. In the four peptides containing D(9k)L synthesized, the poly(Ala) domains retain the ability to undergo a conformational transition from alpha-helical to beta-sheet in (A)(12)-D(9k)L despite the presence of the D(9k)L domain at the center of the peptide molecule, but the presence of this domain in the other model peptides synthesized has a marked effect on the conformation of the added silk-like domains. The results showed that the structures of the Ala-Gly repeating regions can be controlled by the choice of both the organic solvent and the amino acid sequence of the Ala-Gly repeating regions without disrupting the secondary structure of D(9k)L suggesting that it may retain its ability to bind calcium ions.

To obtain detailed structural information for spider dragline spidroin (MaSp1), we prepared three versions of the consensus peptide GGLGGQGAGAAAAAAGGAGQGGYGGLGSQGAGR labeled with 13C at six different sites. The 13C CP/MAS NMR spectra were observed after treating the peptides with different reagents known to alter silk protein conformations. The conformation-dependent 13C NMR chemical shifts and peak deconvolution were used to determine the local structure and the fractional compositions of the conformations, respectively. After trifluoroacetic acid (solvent)/diethyl ether (coagulant) treatment, the N-terminal region of poly-Ala (PLA) sequence, Ala8 and Ala10, adopted predominantly the alpha-helix with a substantial amount of beta-sheet. The central region, Ala15, Ala18, and Leu26, and C-terminal region, Ala31, of the peptide were dominated by either 3(1)-helix or alpha-helix. There was no indication of beta-sheet, although peak broadening indicates that the torsion angle distribution is relatively large. After 9 M LiBr/dialysis treatment, three kinds of conformation, beta-sheet, random coil, and 3(1)-helix, appeared, in almost equal amounts of beta-sheet and random coil conformations for Ala8 and Ala10 residues and distorted 3(1)-helix at the central region of the peptide. In contrast, after formic acid/methanol and 8 M urea/acetonitrile treatments, all of the local structure tends to beta-sheet, although small amounts of random coil are also observed. The peak pattern of the Ala Cbeta carbon after 8 M urea/acetonitrile treatment is similar to the corresponding patterns of silk fiber from Bombyx mori and Samia cynthia ricini. We also synthesized a longer 13C-labeled peptide containing two PLA blocks and three Gly-rich blocks. After 8 M urea/acetonitrile treatment, the conformation pattern was closely similar to that of the shorter peptide.

To avoid potential ambiguity in the structural determination of native spider silk, we prepared both a nonlabeled peptide with a sequence containing both the polyalanine and the repeated GGA region...

Orientation of the Antimicrobial Peptide, Cecropin A–Magainin 2 Hybrid, in a Lipid Bilayer Studied by 15 N Solid-State NMR