Amount Of Β-sheets Research Articles

Biophysical characterization of G protein ectodomain of group B human respiratory syncytial virus from E. coli

ABSTRACTHuman respiratory syncytial virus (hRSV) is an important pathogen of acute respiratory tract infection. The G protein of hRSV is a transmembrane glycoprotein that is a neutralizing antigen and is thus a vaccine candidate. In this study, synthetic codon optimized ectodomain G protein [G(ΔTM)] of BA genotype of group B hRSV was cloned, expressed, and characterized using biophysical techniques. The molar absorption coefficient and mean residue ellipticity at 222 nm ([θ]222) of G (ΔTM) was found to be 7950 M−1 cm−1 and −19701.7 deg cm2 dmol-1 respectively. It was concluded that G(ΔTM) mainly consist of α-helix (74.9%) with some amount of β-sheet (4%). The protein was stable up to 85°C without any transition curve. However, heat-induced denaturation of G(ΔTM) resulted in total loss of β-sheet whereas not much change was observed in the α-helix part of the secondary structure. It was concluded that G(ΔTM) is an α-helical protein and it is highly stable at high temperature, but could be easily denatured using high concentrations of GdmCl/urea or acidic condition. This is the first investigation of cloning, expression, and characterization of G(ΔTM) of BA viruses from India. Structural characterization of G protein will assist in drug designing and vaccine development for hRSV.

Effect of high pressure – low temperature treatments on structural characteristics of whey proteins and micellar caseins

In this study, structural changes in micellar caseins and whey proteins due to high pressure – low temperature treatments (HPLT) were investigated and compared to changes caused by high pressure treatments at room temperature. Whey protein isolate (WPI) solutions as well as micellar casein (MC) dispersions and mixtures were treated at 500MPa (pH 7.0 and 5.8) at room temperature, −15°C and −35°C. Surface hydrophobicity and accessible thiol groups remained nearly unchanged after HPLT treatments whereas HP treatments at room temperature caused an unfolding of the WPI, resulting in an increase in surface hydrophobicity and exposure of the thiol groups. For HPLT treatments, distinct changes in the secondary structure (increase in the amount of β-sheets) were observed while the tertiary structure remained unchanged. Large flocs, stabilized by hydrophobic interactions and hydrogen bonds, were formed in casein containing samples due to HPLT treatments. Depending on the pH and the applied HPLT treatment parameters, these interactions differed significantly from the interactions determined in native micelles.

Nanostructural morphology of plasticized wheat gluten and modified potato starch composites: relationship to mechanical and barrier properties.

In the present study, we were able to produce composites of wheat gluten (WG) protein and a novel genetically modified potato starch (MPS) with attractive mechanical and gas barrier properties using extrusion. Characterization of the MPS revealed an altered chain length distribution of the amylopectin fraction and slightly increased amylose content compared to wild type potato starch. WG and MPS of different ratios plasticized with either glycerol or glycerol and water were extruded at 110 and 130 °C. The nanomorphology of the composites showed the MPS having semicrystalline structure of a characteristic lamellar arrangement with an approximately 100 Å period observed by small-angle X-ray scattering and a B-type crystal structure observed by wide-angle X-ray scattering analysis. WG has a structure resembling the hexagonal macromolecular arrangement as reported previously in WG films. A larger amount of β-sheets was observed in the samples 70/30 and 30/70 WG-MPS processed at 130 °C with 45% glycerol. Highly polymerized WG protein was found in the samples processed at 130 °C versus 110 °C. Also, greater amounts of WG protein in the blend resulted in greater extensibility (110 °C) and a decrease in both E-modulus and maximum stress at 110 and 130 °C, respectively. Under ambient conditions the WG-MPS composite (70/30) with 45% glycerol showed excellent gas barrier properties to be further explored in multilayer film packaging applications.

Metal ion mediated transition from random coil to β-sheet and aggregation of Bri2-23, a natural inhibitor of Aβ aggregation.

Furin-dependent maturation of the BRI2 protein generates the Bri2-23 fragment that is able to arrest the aggregation of amyloidβ, the peptide implicated in Alzheimer's disease (AD). Bri2-23 contains cysteines at positions 5 and 22, which are likely to bind to metal ions such as Cu(i). Metal ions may play a role in the etiology of neurodegenerative disorders such as AD, and in this work we explore the metal ion induced folding and aggregation of Bri2-23 using Hg(ii) and Ag(i) as spectroscopic probes with structural and ligand preferences similar to those of Cu(i), while not displaying redox activity under the experimental conditions. In general, interaction of Bri2-23 with soft metal ions changes the structural properties and solution behavior of the peptide that tune to increasing metal to peptide stoichiometry. Potentiometric, (199m)Hg PAC and ESI-MS data indicate that addition of up to 0.5 equivalents of Hg(ii) to Bri2-23 yields a two-coordinated HgS2 structure at the metal site. While the free peptide is inherently unstructured, the presence of Ag(i) and Hg(ii) gives rise to β-sheet formation. NMR spectroscopy supports the formation of β-sheet structure in the presence of 0.5 equivalents of Hg(ii), and displays an interesting and marked change in the TOCSY spectra when increasing the Hg(ii) to peptide stoichiometry from 0.5 to 0.7 equivalents, indicating the equilibrium between two structural analogues of the complex. Addition of more than 0.7 equivalents of Hg(ii) gives rise to line broadening, presumably reflecting aggregation. This is further supported by ThT fluorescence studies showing that the Bri2-23 peptide does not aggregate over 24 hours, while addition of over 0.7 equivalents of Ag(i) or Hg(ii) leads to increase of fluorescence, indicating that these metal ions induce aggregation. Thus, a model integrating all data into a coherent picture is that the metal ion binding to the two thiolates gives rise to folding of the peptide into a structure that is prone to aggregation, forming aggregates with a considerable amount of β-sheets. Molecular dynamics simulations initiated with structures that agree with NMR data additionally support this model.

Processing and characterization of powdered silk micro- and nanofibers by ultrasonication.

Silk derived from Bombyx mori silkworm cocoons was degummed in an aqueous sodium carbonate solution, and the resulting silk fibroin fibers were placed in an acidic aqueous solution and were treated with ultrasonication to obtain powdered micro- and nanofibers. The morphologies and spectral characteristics of these powdered silk fibers were investigated in detail. The shape, surface and structural features of the powdered fibers were affected by the ultrasonic power and media. Increasing the acidity of the ultrasonic solution and increasing the ultrasonic power increased the fiber breakage speed, resulting in shorter fiber lengths. Powdered microfibers could not be obtained in a formic acid solution, while powdered nanofibers whose diameter below 1μm were obtained in a combined formic acid and hydrochloric acid ultrasonication solution. Observation via SEM and optical microscopy revealed that the microfiber diameters were approximately 5-10μm, and those of the nanofibers were approximately 30-120nm. The analysis of laser sizer showed that the microfiber sizes ranged mainly from 20 to 100μm. FT-IR and XRD spectra demonstrated that the relative amount of β-sheets increased after the ultrasonic treatment. The ε-amino group content on the surface of the micro- and nanofibers increased significantly. These studies provide reliable methods for the preparation of nano-scale silk fibroin fibers by ultrasonication and open new avenues for the development of powdered silk fibers as advanced functional biomaterials.

Human Lactoferricin Derivatives as New Targeted Weapons in Cancer Therapy

In our study derivatives of a short cationic peptide derived from the human host defense peptide Lactoferricin (LFcin) were optimized in their activity and selectivity towards cancer cells. The negatively charged membrane lipid phosphatidylserine (PS) serves as a target for these peptides since PS is specifically exposed by cancer cells (1).Calorimetric and permeability studies showed that N-acylation and even more the repeat sequences of derivatives of hLFcin leads to strongly improved interaction with the cancer mimic PS, whereas the healthy mimic phosphatidylcholine (PC) is only slightly affected by the lipopeptide. Tryptophan fluorescence of selective peptides revealed peptide penetration only into the PS membrane interface and circular dichroism showed change of structure by increase of amount of β-sheets just in the presence of the cancer mimic. We demonstrated that hLFcin derivatives also exhibit anticancer activity in vitro against several cancer cell lines, correlating with selective activity against the cancer model PS. Active and selective peptides induced apoptosis only in cancer cells, whereas melanocytes and fibroblasts remained unaffected at same concentrations, yielding specificity for cancer cells higher than 100-fold for some peptides. Currently, first in vivo studies are under progress.The data indicate the need of high affinity to the target PS, a minimum length and positive net charge, an adequate but moderate hydrophobicity, and capability of adoption of a defined structure exclusively in presence of the target membrane for high antitumor activity.Acknowledgment: Austrian Science Foundation FWF (grant no. P20760, P24608)(1) Riedl et al. BBA 1808: 2638-2645, 2011.

Conformation and Activity of Glucose Oxidase on Homogeneously Coated and Nanostructured Surfaces

Protein unfolding and loss of protein function upon surface contact is a major problem in biotechnology and biomedicine. Using glucose oxidase (GOx) as a model protein, we investigated the impact of surface chemistry, topography, and confinement on enzyme activity, conformation, and affinity. A particular focus lay on the question whether the conformation of surface-bound proteins can be stabilized by embedding nanoscale adsorption sites, here in the form of monodisperse gold nanoparticles (AuNPs), into a protein-repelling matrix material. It was found that on homogeneous surfaces, GOx activity is generally lower than that in its native state and strongly affected by surface chemistry. Loss of activity is related to an increasing amount of β-sheets in the GOx secondary structure and a corresponding reduction of α-helical elements. In contrast, on AuNP surfaces, the effect of surface chemistry is negligible, and the amount of adsorbed protein only depends on particle size. The low activity of GOx on all nanostructures studied is again accompanied by an increase of β-sheet and a reduction of α-helical secondary structure. The major cause for protein unfolding on AuNPs thus seems to be the curvature of the surface. In addition, the data suggest that unfavorable orientation of the adsorbed enzyme also contributes to the loss of activity.

Purification and properties of carboxymethylcellulase (CMCase) from a cellulose-decomposing bacterium Sporocytophaga sp. JL-01

Sporocytophaga sp., a Gram-negative aerobic cellulose decomposing bacterium, is capable of gliding on the surface of solid medium. A new endoglucanase, carboxymethylcellulase (CMCase1), was purified from an aerobic bacteriumSporocytophaga sp. JL-01. The molecular weight of the enzyme was 82 kDa as determined by SDS Polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH was 5.0 and optimum temperature was 50°C under experiment conditions. The Km of CMCase1 was 9 mgml-1 and Vmax was 27.3 ug min-1mg-1 protein. The Zn2+ could inhibit the activity of this enzyme at ion concentration of 5 mM. RP-HPLC revealed that aromatic amino acid, acidic amino acid, and basic amino acid accounted for 10.5, 24.5 and 7.0%, respectively. The far-UV circular dichroism (CD) spectrum showed that the amount of β-sheet was decreased with the temperature changed from 25 to 75°C. Key words: Sporocytophaga, carboxymethylcellulase, enzyme characteristics, circular dichroism spectrum

Structure, Stability, and Interaction of Fibrin αC-Domain Polymers

Our previous studies revealed that in fibrinogen the αC-domains are not reactive with their ligands, suggesting that their binding sites are cryptic and become exposed upon its conversion to fibrin, in which these domains form αC polymers. On the basis of this finding, we hypothesized that polymerization of the αC-domains in fibrin results in the exposure of their binding sites and that these domains adopt the physiologically active conformation only in αC-domain polymers. To test this hypothesis, we prepared a recombinant αC region (residues Aα221-610) including the αC-domain (Aα392-610), demonstrated that it forms soluble oligomers in a concentration-dependent and reversible manner, and stabilized such oligomers by covalently cross-linking them with factor XIIIa. Cross-linked Aα221-610 oligomers were stable in solution and appeared as ordered linear, branching filaments when analyzed by electron microscopy. Spectral studies revealed that the αC-domains in such oligomers were folded into compact structures of high thermal stability with a significant amount of β-sheets. These findings indicate that cross-linked Aα221-610 oligomers are highly ordered and mimic the structure of fibrin αC polymers. The oligomers also exhibited functional properties of polymeric fibrin because, in contrast to the monomeric αC-domain, they bound tPA and plasminogen and stimulated activation of the latter by the former. Altogether, the results obtained with cross-linked Aα221-610 oligomers clarify the structure of the αC-domains in fibrin αC polymers and confirm our hypothesis that their binding sites are exposed upon polymerization. Such oligomers represent a stable, soluble model of fibrin αC polymers that can be used for further structure-function studies of fibrin αC-domains.

When a module is not a domain: the case of the REJ module and the redefinition of the architecture of polycystin-1.

The extracellular region of a group of cell-surface receptors known as the polycystic kidney disease 1 family, containing, among others, polycystin-1, has been controversially described as containing four FNIII (fibronectin typeIII) domains or one REJ (receptor of egg jelly protein) module in the same portion of polypeptide. Stimulated by recent atomic force microscopy work, we re-examined the similarity of these four domains with a FNIII sequence profile showing the evolutionary relationship. Two of the predicted domains could be expressed in bacteria and refolded to give a protein suitable for biophysical study, and one of these expressed solubly. CD spectroscopy showed that both domains contain a significant amount of β-sheet, in good agreement with theoretical predictions. Confirmation of independent folding as a domain is obtained from highly co-operative thermal and urea unfolding curves. Excellent dispersion of peaks in the high-field region of one-dimensional NMR spectra confirms the presence of a hydrophobic core. Analytical ultracentrifugation and analytical gel filtration agree very well with the narrow linewidths in the NMR spectra that at least one of the domains is monomeric. On the basis of this combined theoretical and experimental analysis, we show that the extracellular portion of polycystin-1 does indeed contain β-sheet domains, probably FNIII, and that, consequently, the REJ module is not a single domain.

Protein network structure and properties of wheat gluten extrudates using a novel solvent-free approach with urea as a combined denaturant and plasticiser

This is, to our knowledge, the first success on solvent-free extrusion of wheat gluten (WG) into high quality films without using NaOH/salicylic acid. It was possible by using urea (concentrations: 10, 15 and 20 wt%) in the single screw-extruder process. Tensile testing, oxygen permeability, water vapor transmission rate, infrared spectroscopy (IR), confocal laser scanning microscopy (CLSM) and protein solubility were used to assess the properties of the extrudates. As the urea concentration increased, the strength and stiffness decreased while the extensibility increased. The oxygen permeability was low and increased, as did the water vapor transmission rate, with increasing urea concentration. The protein solubility of urea-containing films was found to be significantly lower than that of the native gluten and glycerol-plasticized WG extrudate. CLSM, together with the protein solubility, indicated that the urea films were aggregated/polymerized and IR spectroscopy revealed that these films contained a sizeable amount of β-sheets with a high degree of hydrogen bonds associated with protein aggregation. The aggregation did not change with increasing urea concentration, which suggests that the changes in the mechanical and permeability properties were due to urea-induced plasticisation.

Structural and conformational modification of whey proteins induced by supercritical carbon dioxide

Abstract In this paper, the effect of supercritical carbon dioxide (SC-CO2) treatment on the structure and conformation of whey protein isolate (WPI) were investigated. The WPI solution treated with SC-CO2 at 20 MPa and 30–60 °C for 1 h showed that its turbidity and particle size were increased. The effect of SC-CO2 treatment at 60 °C on the fluorescence intensity of WPI was elevated indicating partial denaturation of its fractions and exposure of more hydrophobic regions. The secondary structure change of SC-CO2 treated WPI revealed a decrease in the α-helix content, hydrogen bonds and an increase in the amount of β-sheet. A high shift of endothermic peak after SC-CO2 treatment was observed by the thermal analysis of WPI. These results confirmed that the structure and conformation of protein were modified with SC-CO2 treatment. Industrial relevance There is a strong interest in the food industry in studying the modifying protein functionality to produce higher quality products. Supercritical carbon dioxide (SC-CO2) treatment described as green method has raised interests in its application for polymer modification. SC-CO2 is a unique processing method offering many advantages over conventional thermal processing methods, including the ability to retain the quality of food, which may be lost during conventional thermal processing. This work explores the SC-CO2 effect on the structural and conformational modification of protein which would be extremely helpful in understanding the improving of the protein functionalities such as, emulsifying and rheological properties in order to produce higher quality food products.

Silk gland sericin protein membranes: Fabrication and characterization for potential biotechnological applications

This study describes the potential use of silk gland sericin protein as a biocompatible natural biopolymer in its native form. The membranes were fabricated using native silk sericin protein extracted from middle silk gland of Antheraea mylitta, a non-mulberry tropical tasar silkworm without using any cross-linking agent. The fabricated membranes were biophysically characterized and optimized for cell culture. Silk sericin protein extracted from gland contained higher amount of β-sheets, which increased upon treatment with ethanol as observed by FTIR and XRD. The membranes did show robustness, good mechanical strength and high temperature stability. Cytocompatibility of the membranes was evaluated by MTT assay and cell cycle analysis using feline fibroblast cells. Morphology of growing cells was assessed by confocal microscopy that indicated normal spreading and proliferation on the silk sericin membranes. The membranes showed low inflammatory response as observed assaying TNF α release. This study reveals the potential of native silk sericin protein from silk gland as biocompatible biopolymer for potential biomedical applications.

Influence of Residue 22 on the Folding, Aggregation Profile, and Toxicity of the Alzheimer's Amyloid β Peptide

Several biophysical techniques have been used to determine differences in the aggregation profile (i.e., the secondary structure, aggregation propensity, dynamics, and morphology of amyloid structures) and the effects on cell viability of three variants of the amyloid β peptide involved in Alzheimer's disease. We focused our study on the Glu22 residue, comparing the effects of freshly prepared samples and samples aged for at least 20 days. In the aged samples, a high propensity for aggregation and β-sheet secondary structure appears when residue 22 is capable of establishing polar (Glu22 in wild-type) or hydrophobic (Val22 in E22V) interactions. The Arctic variant (E22G) presents a mixture of mostly disordered and α-helix structures (with low β-sheet contribution). Analysis of transmission electron micrographs and atomic force microscopy images of the peptide variants after aging showed significant quantitative and qualitative differences in the morphology of the formed aggregates. The effect on human neuroblastoma cells of these Aβ12–28 variants does not correlate with the amount of β-sheet of the aggregates. In samples allowed to age, the native sequence was found to have an insignificant effect on cell viability, whereas the Arctic variant (E22G), the E22V variant, and the slightly-aggregating control (F19G-F20G) had more prominent effects.

Attenuated Total Reflection Infrared Spectroscopy: An Efficient Technique to Quantitatively Determine the Orientation and Conformation of Proteins in Single Silk Fibers

Polarized attenuated total reflection (ATR) infrared spectroscopy is an efficient technique to determine the orientation and conformation of a large variety of samples, but it is more difficult to apply to very small specimens such as silk fibers. The Golden Gate single-reflection ATR accessory that uses diamond as an ATR element and a focalized beam turns out to be highly efficient to study quantitatively the orientation and conformation of a single silk fibroin filament of the silkworm Bombyx mori that is about 10 mum in diameter. For orientation measurements, rotating the sample instead of the electric field greatly simplifies the theoretical analysis and keeps the penetration depth of the infrared radiation constant. A sample holder that can be fitted on the ATR accessory has thus been developed to allow accurate rotation of the sample and to obtain spectra with a low, non-damaging, and reproducible pressure on the fiber. To validate the method, spectra have been recorded as a function of the angle theta between the fiber axis and the polarization of the incident radiation. The data have been fitted following the cosine square dependency of the absorbance with respect to the angle theta. The procedure has been applied to the spectral components of the amide I bands, as determined from spectral decomposition. Multiple angle measurements turn out to be quite useful to correct systematic angle errors and validate the accuracy of the curve-fitting parameters of the band decomposition. By using the calculated dichroic ratio, a parameter <P2> of -0.46+/-0.01 has been calculated for the antiparallel beta-sheets and -0.04+/-0.02 for the remaining structures. From the orientation-insensitive spectrum A0, the amount of beta-sheets has been estimated to 49+/-3%. The results obtained from only two measurements with the electric field of the incident radiation parallel and perpendicular to the fiber axis has demonstrated that ATR spectroscopy can be used routinely in quantitative studies of the molecular orientation and conformation of macromolecules.

Thermoreversible lysozyme hydrogels: properties and an insight into the gelation pathway.

The gelation behaviour of aqueous solutions of hen egg white lysozyme (HEWL) in the presence of 20 mM DTT in the concentration range 0.7 to 4.0 mM has been investigated using microDSC, FTIR, cryoTEM, SANS and oscillatory rheology. The macroscopic critical gelation concentration, Cgel, was found to be ∼ 3.0 mM. The disruption of the disulfide bonds by the DTT and the destabilisation of the protein were found to be a prerequisite for the formation of β-sheet rich fibrils under the mild conditions used in this work. Using our methodology the hydrogels obtained have a pH of 7, hence are suitable for cell culture, and are also thermoreversible. The hydrogel melting temperature was found to increase with increasing concentration and a similar structure was observed across the concentration range investigated. Our results suggest these systems are composed of a well defined regular network where single β-sheet rich fibrils (∼ 3 nm diameter) form initially, then two of these fibrils associate two-by-two to form junctions (∼ 6 nm diameter) and then on cooling further aggregate to form larger bundles of fibres. The network mesh size was found to decrease with increasing concentration. Our results suggest that below Cgel small unconnected gel-like aggregates exist that have a similar structure to the hydrogels obtained above Cgel. Using our data we propose a model for the denaturation and gelation behaviour of our system. During the first heating an α-helix to β-sheet molecular transition for the protein conformation occurs resulting in β-sheet rich fibrils forming through the self-assembly of β-sheet rich denaturated proteins. At high temperature the solution contains β-sheet rich fibrils with dissolved protein. On cooling an increase in the amount of β-sheet was observed via FTIR suggesting that as the temperature is decreased more and more protein forms β-sheet rich fibrils. At the gelation temperature these fibrils associate two-by-two to form the network junctions resulting in the macroscopic gelation of the sample. Our results suggest the network junctions are formed via specific hydrophobic interactions. The hydrogels elastic modulus was found to scale as C2.45 for C > Cgel.

Identification and characterization of a novel toxin–antitoxin module from Bacillus anthracis

Comparative genome analysis of Bacillus anthracis revealed a pair of linked genes encoding pemK (K, killer protein) and pemI (I, inhibitory protein) homologous to pem loci of other organisms. Expression of PemK in Escherichia coli and Bacillus anthracis was bacteriostatic whereas the concomitant expression of PemI reversed the growth arrest. PemK expression effectively inhibited protein synthesis with no significant effect on DNA replication. Coexpression and interaction of these proteins confirmed it to be a Type II addiction module. Thermal denaturation analysis reflected poor conformational stability of PemI as compared to PemK. Circular dichroism analysis indicated that PemI contains twice the amount of β-sheets as PemK. Gel retardation assays demonstrated that PemI binds to its upstream DNA sequence. This study reports the first evidence of an active chromosome encoded toxin–antitoxin locus in B. anthracis.

Characterization by Raman Microspectroscopy of the Strain-Induced Conformational Transition in Fibroin Fibers from the Silkworm Samia cynthia ricini

Raman microspectroscopy has been used to quantitatively study the effect of a mechanical deformation on the conformation and orientation of Samia cynthia ricini (S. c. ricini) silk fibroin. Samples were obtained from the aqueous solution stored in the silk gland and stretched at draw ratios (lambda) ranging from 0 to 11. Using an appropriate band decomposition procedure, polarized and orientation-insensitive spectra have been analyzed to determine order parameters and the content of secondary structures, respectively. The data unambiguously show that, in response to mechanical deformation, S. c. ricini fibroin undergoes a cooperative alpha-helix to beta-sheet conformational transition above a critical draw ratio of 4. The alpha-helix content decreases from 33 to 13% when lambda increases from 0 to 11, while the amount of beta-sheets increases from 15 to 37%. In comparison, cocoon silk is devoid of alpha-helical structure and always contains a larger amount of beta-sheets. Although the presence of isosbestic points in different spectral regions reveals that the conformational change induced by mechanical deformation is a two-state process, our results suggest that part of the glycine residues might be incorporated into beta-poly(alanine) structures. The beta-sheets are initially isotropically distributed and orient along the fiber axis as lambda increases, but do not reach the high level of orientation found in the cocoon fiber. The increase in the orientation level of the beta-sheets is found to be concomitant with the alpha --> beta conformational conversion, whereas alpha-helices do not orient under the applied strain but are rather readily converted into beta-sheets. The components assigned to turns exhibit a small orientation perpendicular to the fiber axis in stretched samples, showing that, overall, the polypeptide chains are aligned along the stretching direction. Our results suggest that, in nature, factors other than stretching contribute to the optimization of the amount of beta-sheets and the high degree of orientation found in natural cocoon silk.

Structure of Model Peptides Based on Nephila clavipes Dragline Silk Spidroin (MaSp1) Studied by 13C Cross Polarization/Magic Angle Spinning NMR

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.

Seeding-dependent Propagation and Maturation of Amyloid Fibril Conformation

Recent studies of amyloid fibrils have focused on the presence of multiple amyloid forms even with one protein and their propagation by seeding, leading to conformational memory. To establish the structural basis of these critical features of amyloid fibrils, we used the amyloidogenic fragment Ser20-Lys41 (K3) of beta2-microglobulin, a protein responsible for dialysis-related amyloidosis. In 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH approximately 2), K3 peptide formed two types of amyloid-like fibrils, f218 and f210, differing in the amount of beta-sheet as measured by circular dichroism spectroscopy and Fourier transform infrared spectroscopy. Atomic force microscopy showed that the fibril with a larger amount of beta-sheet (f210) is thinner and longer. Both fibrils were reproduced by seeding, showing the template-dependent propagation of a fibril's conformation. However, upon repeated self-seeding, f218 fibrils were gradually transformed into f210 fibrils, revealing the conformational maturation. The observed maturation can be explained fully by a competitive propagation of two fibrils. The maturation of amyloid fibrils might play a role during the development of amyloidosis.