Antiparallel Β-pleated Sheet Research Articles

Crystal structure and physical properties of Antheraea yamamai silk fibers: Long poly(alanine) sequences are partially in the crystalline region

Antheraea yamamai silk contains long poly(alanine) sequences that contribute to β-sheet crystals, which are responsible for the high tensile strength of silk fibers. We determined the crystal structure, physical properties, and morphology of A. yamamai cocoon silk fibers. The crystal lattice of A. yamamai silk fiber consists of antiparallel β-pleated sheets with an orthogonal unit cell (a = 10.72 Å, b = 9.73 Å, c [fiber axis] = 6.80 ± 0.05 Å). Wide-angle X-ray scattering and birefringence measurements revealed that stretching deformation does not affect the crystal structure but contributes to alignment of silk molecules along the fiber axis in the partial amorphous phase. Crystallinity and amino acid sequence analyses suggested that the poly(alanine) region can be partially crystallized. The long poly(alanine) sequences therefore do not contribute to the mechanical, thermal, or structural properties of A. yamamai silk. Our results may aid the design and development of A. yamamai silk-based materials.

Thiazole‐Based γ‐Building Blocks as Reverse‐Turn Mimetic to Design a Gramicidin S Analogue: Conformational and Biological Evaluation

This paper describes the ability of a new class of heterocyclic γ-amino acids named ATCs (4-amino(methyl)-1,3-thiazole-5-carboxylic acids) to induce turns when included in a tetrapeptide template. Both hybrid Ac-Val-(R or S)-ATC-Ile-Ala-NH2 sequences were synthesized and their conformations were studied by circular dichroism, NMR spectroscopy, MD simulations, and DFT calculations. It was demonstrated that the ATCs induced highly stable C9 pseudocycles in both compounds promoting a twist turn and a reverse turn conformation depending on their absolute configurations. As a proof of concept, a bioactive analogue of gramicidin S was successfully designed using an ATC building block as a turn inducer. The NMR solution structure of the analogue adopted an antiparallel β-pleated sheet conformation similar to that of the natural compound. The hybrid α,γ-cyclopeptide exhibited significant reduced haemotoxicity compared to gramicidin S, while maintaining strong antibacterial activity.

Retracted Article: Determination of silk fibroin secondary structure by terahertz time domain spectroscopy

Silk fibroin membranes with different secondary structures were fabricated, and subsequently employed as an ideal system model for the investigation into the conformational transition of native proteins by terahertz time domain spectroscopy (THz-TDS). In comparison to conventional Fourier Transform Infrared (FTIR) Spectroscopy, THz spectroscopy presents distinct features for each kind of secondary structure with narrower bandwidths. Based on the theoretical calculation, absorption features observed in the range of 2.0–2.6 THz may be attributed to intramolecular modes of peptide chain. Three bands centered at 1.84 THz, 1.68 THz, and 1.55 THz are attributed to random coil, α-helix, and antiparallel β-pleated sheet, respectively. The results indicate that THz-TDS presents great potential as a complementary method to FTIR in determining the secondary structure of silk fibroin.

Preparation and characterization of silk fibroin as a biomaterial with potential for drug delivery.

BackgroundDegummed silk fibroin from Bombyx mori (silkworm) has potential carrier capabilities for drug delivery in humans; however, the processing methods have yet to be comparatively analyzed to determine the differential effects on the silk protein properties, including crystalline structure and activity.MethodsIn this study, we treated degummed silk with four kinds of calcium-alcohol solutions, and performed secondary structure measurements and enzyme activity test to distinguish the differences between the regenerated fibroins and degummed silk fibroin.ResultsGel electrophoresis analysis revealed that Ca(NO3)2-methanol, Ca(NO3)2-ethanol, or CaCl2-methanol treatments produced more lower molecular weights of silk fibroin than CaCl2-ethanol. X-ray diffraction and Fourier-transform infrared spectroscopy showed that CaCl2-ethanol produced a crystalline structure with more silk I (α-form, type II β-turn), while the other treatments produced more silk II (β-form, anti-parallel β-pleated sheet). Solid-State 13C cross polarization and magic angle spinning-nuclear magnetic resonance measurements suggested that regenerated fibroins from CaCl2-ethanol were nearly identical to degummed silk fibroin, while the other treatments produced fibroins with significantly different chemical shifts. Finally, enzyme activity test indicated that silk fibroins from CaCl2-ethanol had higher activity when linked to a known chemotherapeutic drug, L-asparaginase, than the fibroins from other treatments.ConclusionsCollectively, these results suggest that the CaCl2-ethanol processing method produces silk fibroin with biomaterial properties that are appropriate for drug delivery.

Integrity of Helix 2-Helix 3 Domain of the PrP Protein Is Not Mandatory for Prion Replication

The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrP(Sc). We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrP(Sc) and elucidate the conformational changes underlying prions generation.

The role of entropy in initializing the aggregation of peptides: a first principle study on oligopeptide oligomerization

The initiation and progression of Alzheimer's disease is coupled to the oligo- and polymerization of amyloid peptides in the brain. Amyloid like aggregates of protein domains were found practically independent of their primary sequences. Thus, the driving force of the transformation from the original to a disordered amyloid fold is expected to lie in the protein backbone common to all proteins. In order to investigate the thermodynamics of oligomerization, full geometry optimizations and frequency calculations were performed both on parallel and antiparallel β-pleated sheet model structures of [HCO-(Ala)(1-6)-NH(2)](2) and (For-Ala(1-2)-NH(2))(1-6) peptides, both at the B3LYP and M05-2X/6-311++G(d,p)//M05-2X/6-31G(d) levels of theory, both in vacuum and in water. Our results show that relative entropy and enthalpy both show a hyperbolic decrease with increasing residue number and with increasing number of strands as well. Thus, di- and oligomerization are always thermodynamically favored. Antiparallel arrangements were found to have greater stability than parallel arrangements of the polypeptide backbones. During our study the relative changes in thermodynamic functions are found to be constant for long enough peptides, indicating that stability and entropy terms are predictable. All thermodynamic functions of antiparallel di- and oligomers show a staggered nature along the increasing residue number. By identifying and analyzing the 6 newly emerging dimer vibrational modes of the 10- and 14-membered building units, the staggered nature of the entropy function can be rationalized. Thus, the vanishing rotational and translational modes with respect to single strands are converted into entropy terms "holding tight" the dimers and oligomers formed, rationalizing the intrinsic adherence of natural polypeptide backbones to aggregate.

Macro/microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications

This study describes the developmental physicochemical properties of silk fibroin scaffolds derived from high-concentration aqueous silk fibroin solutions. The silk fibroin scaffolds were prepared with different initial concentrations (8, 10, 12 and 16%, in wt.%) and obtained by combining the salt-leaching and freeze-drying methodologies. The results indicated that the antiparallel β-pleated sheet (silk-II) conformation was present in the silk fibroin scaffolds. All the scaffolds possessed a macro/microporous structure. Homogeneous porosity distribution was achieved in all the groups of samples. As the silk fibroin concentration increased from 8 to 16%, the mean porosity decreased from 90.8±0.9 to 79.8±0.3% and the mean interconnectivity decreased from 97.4±0.5 to 92.3±1.3%. The mechanical properties of the scaffolds exhibited concentration dependence. The dry state compressive modulus increased from 0.81±0.29 to 15.14±1.70MPa and the wet state dynamic storage modulus increased by around 20- to 30-fold at each testing frequency when the silk fibroin concentration increased from 8 to 16%. The water uptake ratio decreased with increasing silk fibroin concentration. The scaffolds present favorable stability as their structure integrity, morphology and mechanical properties were maintained after in vitro degradation for 30days. Based on these results, the scaffolds developed in this study are proposed to be suitable for use in meniscus and cartilage tissue-engineered scaffolding.

Secondary structure of chorion proteins of the Lepidoptera Pericallia ricini and Ariadne merione by ATR FT-IR and micro-Raman spectroscopy

The gross morphological features of the eggs and eggshells (chorions) of two Lepidoptera species, Pericallia ricini and Ariadne merione were revealed for the first time by scanning and transmission electron microscopy. These two insect pests are extremely serious threats for many crops, mainly in India, but also in several other regions of the world. Micro-Raman and ATR FT-IR spectroscopy were also applied to study in detail the secondary structure of the eggshell (chorion) proteins of these Lepidoptera species. Both techniques indicate that the two species have nearly identical conformations of their chorion proteins with abundant antiparallel β-pleated sheet. These results are in support of our previous findings that the helicoidal architecture of the proteinaceous chorion of Lepidoptera and fishes is dictated by a common molecular denominator, the antiparallel β-pleated sheet secondary structure.

Isolation and characterization of SAP and CRP, two pentraxins from Pangasianodon ( Pangasius) hypophthalmus

From the serum of Pangasianodon hypophthalmus, two proteins were isolated by affinity chromatography on Sepharose and phosphorylcholine–Sepharose. Their binding on the affinity matrices critically depends on the presence of Ca 2+ ions. N-terminal sequencing and sequencing of internal tryptic peptides identified the proteins as pentraxins and from their binding properties they are identified as SAP (serum amyloid P component) and CRP (C-reactive protein). Per ml serum, 36 μg SAP and 56 μg CRP was purified. Upon gel filtration, both the SAP and CRP elute as trimers of respectively 24 kDa and 28 kDa subunits. Both proteins are devoid of inter-chain disulfide bonds. Both SAP and CRP are glycosylated and agglutinate rabbit erythrocytes and pathogenic bacteria Edwardsiella ictaluri and Aeromonas hydrophila, but not Micrococcus lysodeikticus or Escherichia coli. Haemagglutination of SAP and CRP is inhibited by galactose (MIC = 1 mM) and by phosphorylcholine (MIC = 1–2 mM), respectively. Circular dichroism studies revealed that antiparallel β-pleated sheets are dominating the secondary structure. Upon removing the Ca 2+ ions by EDTA, slight structural changes are observed by CD spectroscopy in the near-UV region. Immunodiffusion shows that P. hypophthalmus SAP and CRP do not cross-react.

Crystal Structure of the Novel Complex Formed between Zinc α2-Glycoprotein (ZAG) and Prolactin-Inducible Protein (PIP) from Human Seminal Plasma

This is the first report on the formation of a complex between zinc α2-glycoprotein (ZAG) and prolactin-inducible protein (PIP). The complex was purified from human seminal plasma and crystallized using 20% polyethylene glycol 9000 and 5% hexaethylene glycol. The structure of the complex has been determined using X-ray crystallographic method and refined to an Rcryst of 0.199 (Rfree=0.239). The structure of ZAG is broadly similar to the structure of serum ZAG. The scaffolding of PIP consists of seven β-strands that are organized in the form of two antiparallel β-pleated sheets, resulting in the formation of a sandwiched β-sheet. The amino acid sequence of PIP contains one potential N-glycosylation site at Asn77, and the same is found glycosylated with four sugar residues. The structure of the complex shows that the β-structure of PIP is ideally aligned with the β-structure of domain α3 of ZAG to form a long interface between two proteins. The proximal β-strands at the long interface are arranged in an antiparallel manner. There are 12 hydrogen bonds and three salt bridges between ZAG and PIP. At the two ends of vertical interface, two salt bridges are formed between pairs of Lys41–Asp233 and Lys68–Glu229. On the perpendicular interface involving α1–α2 domains of ZAG and a loop of PIP, another salt bridge is formed. The internal space at the corner of the L-shaped structure is filled with solvent molecules including a carbonate ion. The overall buried area in the complex is approximately 914 Å2, which is considerably higher than the 660 Å2 reported for the class I major histocompatibility complex structures.

Defective Human Leukocyte Antigen Class I-associated Antigen Presentation Caused by a Novel β2-Microglobulin Loss-of-function in Melanoma Cells

The major histocompatibility complex class I molecules consist of three subunits, the 45-kDa heavy chain, the 12-kDa beta(2)-microglobulin (beta(2)m), and an approximately 8-9-residue antigenic peptide. Without beta(2)m, the major histocompatibility complex class I molecules cannot assemble, thereby abolishing their transport to the cell membrane and the subsequent recognition by antigen-specific T cells. Here we report a case of defective antigen presentation caused by the expression of a beta(2)m with a Cys-to-Trp substitution at position 25 (beta(2)m(C25W)). This substitution causes misfolding and degradation of beta(2)m(C25W) but does not result in complete lack of human leukocyte antigen (HLA) class I molecule expression on the surface of melanoma VMM5B cells. Despite HLA class I expression, VMM5B cells are not recognized by HLA class I-restricted, melanoma antigen-specific cytotoxic T lymphocytes even following loading with exogenous peptides or transduction with melanoma antigen-expressing viruses. Lysis of VMM5B cells is restored only following reconstitution with exogenous or endogenous wild-type beta(2)m protein. Together, our results indicate impairment of antigenic peptide presentation because of a dysfunctional beta(2)m and provide a mechanism for the lack of close association between HLA class I expression and susceptibility of tumor cells to cytotoxic T lymphocytes-mediated lysis in malignant diseases.

A molecular dynamics study of the pentacyclo-undecane cage amino acid tripeptide

In this paper, we report on the conformational profile of the pentacyclo-undecane (PCU) cage tripeptide carried out by molecular dynamics (MD) simulation using water as an explicit solvent. The MD solution phase studies carried on the model peptide analogues (A)=Ac–Ala–Ala–Ala–NHMe; (B)=Ac–Cage–Cage–Cage–NHMe; (C)=Ac–Ala–Cage–Ala–NHMe and (D)=Ac–Ala–Pro–Ala–NHMe, are used as a complimentary technique to the corresponding gas phase simulated annealing (SA) study previously carried out in our laboratory. No significant structural changes were observed over the MD trajectories. However, the results reported here provide further evidence that the (PCU) cage amino acid exhibits C 7eq, C 7aq, α R and α L conformations, and the theoretical results suggest that the PCU cage amino acid is a strong β-turn inducer. These results support the prediction that when the PCU cage residues are in the ( i) and ( i+2) positions, the β-turn can be extended in either direction to form anti-parallel β-pleated sheets, thereby forming the basis of the mechanism for the folding back of the chain in a cross-β-turn structure.

A simple and direct isolation of whey components from raw milk by gel filtration chromatography and structural characterization by Fourier transform Raman spectroscopy

A simple and economical method to isolate whey protein from fresh raw milk is developed by serial defatting, casein eliminating, lactose removing, and separating by gel filtration chromatography. Four major whey components, including immunoglobulin (Ig), bovine serum albumin (BSA), β-lactoglobulin (β-Lg) and α-lactalbumin (α-Lac), and a non-protein of low molecular mass (∼1.7 kDa) but strong absorbance at 280 nm, are detected simultaneously. The small non-protein molecule is rich in aromatic amino acids and thiol groups as supported by the structural characterization with near infrared Fourier transform Raman spectroscopy (FT-Raman). FT-Raman results show that the secondary structure of Ig is dominated by anti-parallel β-pleated sheet; BSA is mainly in α-helix; both β-form and unordered structure are important in β-Lg; while α-Lac is mostly in α-helix coupling with random coil. Differences in the Raman profile for each whey component reflect their intrinsic compositional differences and distinct spatial arrangement. The S–S linkages diverging around 510–540 cm −1 indicate that the conformation of disulfide bonds in each whey components is different, which may be responsible for their diversified behaviors in solubility, rheological and functional properties.

Hydrolytic Activity of Amyloid-beta and its Inhibition with Short Peptides

The main component of the amyloid plaque is insoluble Aβ1-42 (Aβ42), which adopts a structure rich in antiparallel β-pleated sheets. Recently, increasing awareness of Aβ intermediates as molten-globule states has paralleled insight into the biological activities of the Aβ conformer. The molten-globule state of Aβ42 displays a less ordered, metastable conformation that is stabilized by the formation of fibrils. The molten-globule state of the protein has many biological properties and understanding the mechanisms of its formation is an important step in devising a therapeutic strategy for Alzheimers disease. There have been many studies of the biological properties of Aβ42 such as self-aggregation, binding to other proteins such as apolipoprotein E, cytotoxicity for neuronal cells, vasoconstriction, oxidative activity with superoxide-mediated singlet-oxygen intermediate and proteolytic activity against casein. Recent studies demonstrated that α-1 anti-chymotrypsin, a member of the serine protease inhibitor (serpin) family is also involved in the amyloid plaque. In this review, we focused on the serine protease-like activity of Aβ42 for casein substrate and the effect of bio-essential metal ions for the activity and also suggest its inhibition with Aβ42 derivatives; Aβ15-22 (QKLVFFAE) which is a potential fragment to prevent Aβ self-aggregation. Consequently, we suggest that the short peptides of this kind may be of use in the therapy of Alzheimers disease. Keywords: alzheimers disease, guanidine hydrochloride, molten-globule, ab peptides, serine protease-like activity, selfaggregation, metal ions, eight-residue derivatives

Amyloid fibrillogenesis of silkmoth chorion protein peptide-analogues via a liquid-crystalline intermediate phase

Chorion, the major component of silkmoth eggshell, consists of the A and B classes of low-molecular weight structural proteins. Chorion protects the oocyte and the developing embryo from environmental hazards and this is due to the extraordinary physical and chemical properties of its constituent proteins. We have shown previously [FEBS Lett. 479 (2000) 141; 499 (2001) 268] that peptide-analogues of the A and B classes of chorion proteins form amyloid fibrils under a variety of conditions, which led us to propose that silkmoth chorion is a natural, protective amyloid. In this work, we present data showing conclusively that, the first main step of amyloid-like fibrillogenesis of chorion peptides is the formation of nuclei of liquid crystalline nature, which is reminiscent of spider-silk formation. We show that these liquid-crystalline nuclei (spherulites) ‘collapse’/deteriorate to form amyloid fibrils in a spectacular manner, important, it seems, for chorion morphogenesis and amyloid fibrillogenesis in general. The molecular ‘switch’ causing this spectacular transformation is, most probably, a conformational transition to the structure of chorion peptides, from a left-handed parallel β-helix to an antiparallel β-pleated sheet. Apparently, these peptides were suitably designed to play this role, after millions of years of molecular evolution.

FT-Raman spectroscopic investigation of lens proteins of tilapia treated with dietary vitamin E

FT-Raman spectroscopy was employed to explore the structural changes of lens proteins in Tilapia lenses affected by dietary vitamin E supplementation. The microenvironment of major lens constituents including thiol compounds, tyrosine, and tryptophan exhibited significant change upon vitamin E treatment, while the protein secondary structure was unaltered and remained as an antiparallel β-pleated sheet. These structures in the cortex were more susceptible to vitamin E treatment than in the nucleus. Protein sulfhydryls in the cortex were predominantly in the reduced form, while in the nucleus both the oxidized and reduced forms coexisted as evidenced by the vibrational mode of SH (2580 cm −1) and SS (507 cm −1), respectively. Both tyrosine and tryptophan were more accessible to water or more exposed in the cortex than in the nucleus. The symmetrically inverse response of vitamin E, between Raman intensity of 1090 cm −1 and the glutathione level, was consistent with a close relationship of GSH and vitamin E in defending the lens from external insults.

Congo Red Populates Partially Unfolded States of an Amyloidogenic Protein to Enhance Aggregation and Amyloid Fibril Formation

Congo red (CR) has been reported to inhibit or enhance amyloid fibril formation by several proteins. To gain insight into the mechanism(s) for these apparently paradoxical effects, we studied as a model amyloidogenic protein, a dimeric immunoglobulin light chain variable domain. With a range of molar ratios of CR, i.e. r = [CR]/[protein dimer], we investigated the aggregation kinetics, conformation, hydrogen-deuterium exchange, and thermal stability of the protein. In addition, we used isothermal titration calorimetry to characterize the thermodynamics of CR binding to the protein. During incubation at 37 degrees C or during thermal scanning, with CR at r = 0.3, 1.3, and 4.8, protein aggregation was greatly accelerated compared with that measured in the absence of the dye. In contrast, with CR at r = 8.8, protein unfolding was favored over aggregation. The aggregates formed with CR at r = 0 or 0.3 were typical amyloid fibrils, but mixtures of amyloid fibrils and amorphous aggregates were formed at r = 1.3 and 4.8. CR decreased the apparent thermal unfolding temperature of the protein. Furthermore, CR perturbed the tertiary structure of the protein without significantly altering its secondary structure. Consistent with this result, CR also increased the rate of hydrogen-deuterium exchange by the protein. Isothermal titration calorimetry showed that CR binding to the protein was enthalpically driven, indicating that binding was mainly the result of electrostatic interactions. Overall, these results demonstrate that at low concentrations, CR binding to the protein favors a structurally perturbed, aggregation-competent species, resulting in acceleration of fibril formation. At high CR concentration, protein unfolding is favored over aggregation, and fibril formation is inhibited. Because low concentrations of CR can promote amyloid fibril formation, the therapeutic utility of this compound or its analogs to inhibit amyloidoses is questionable.

A structural model of the chitin-binding domain of cuticle proteins

The nature of the interaction of insect cuticular proteins and chitin is unknown even though about half of the cuticular proteins sequenced thus far share a consensus region that has been predicted to be the site of chitin binding. We previously predicted the preponderance of β-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). Consequently, we have also verified experimentally the abundance of antiparallel β-pleated sheet in the structure of cuticle proteins (Iconomidou et al., Insect Biochem. Mol. Biol. 31 (2001) 877). In this work, based on sequence and secondary structure similarity of cuticle proteins, and especially that of the consensus motif, to that of bovine plasma retinol binding protein (RBP), we propose by homology modelling an antiparallel β-sheet half-barrel structure as the basic folding motif of cuticle proteins. This folding motif may provide the template for elucidating cuticle protein–chitin interactions in detail and reveal the precise geometrical formation of cuticle’s helicoidal architecture. This predicted motif is another example where nature utilizes an almost flat protein surface covered by aromatic side chains to interact with the polysaccharide chains of chitin.

Intrinsic properties of allergens and environmental exposure as determinants of allergenicity.

Intrinsic properties of allergens and environmental exposure as determinants of allergenicity.

Penetratin-induced aggregation and subsequent dissociation of negatively charged phospholipid vesicles

The interaction of the cellular delivery vector penetratin with a model system consisting of negatively charged phospholipid vesicles has been studied. Above a certain peptide to lipid molar ratio, the cationic oligopeptide induces vesicle aggregation. Interestingly, the aggregation is followed by spontaneous disaggregation, which may be related to membrane translocation of the peptide. Circular dichroism (CD) measurements indicate a conformational transition, from α-helix to antiparallel β-pleated sheet, which is simultaneous with the aggregation process. The potential influence of spectroscopic artifacts on CD data due to the drastically increased turbidity during aggregation is discussed.