Antiparallel Β-pleated Sheet Research Articles

“Soft”-cuticle protein secondary structure as revealed by FT-Raman, ATR FT-IR and CD spectroscopy

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 a 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). In this study, we examined experimentally the secondary structure of intact and guanidine hydrochloride extracted cuticle and the cuticular protein extract. The studied cuticle came from the larval dorsal abdomen of the lepidopteran Hyalophora cecropia, a classical example of “soft” cuticle. Analysis with FT-Raman, ATR FT-IR and CD spectroscopy indicates that antiparallel β-pleated sheet is the predominant molecular conformation of “soft-cuticle” proteins both in situ in the cuticle and following extraction. It seems that this conformation dictates the modes of chitin–protein interaction in cuticle, in agreement with earlier proposals (Atkins, J. Biosci. 8 (1985) 375).

Computational studies on the binding of β-sheet breaker (BSB) peptides on amyloid βA(1–42)

Alzheimer's disease starts with the aggregation of β-amyloid peptides overproduced in the brain. The pathognomic plaques contain 50–100 peptides in parallel and/or antiparallel β-pleated sheet structure. Short peptide fragments called β-sheet breaker (BSB) peptides can bind specifically to β-amyloid peptides hindering the association and aggregation. The 3D structures of the molecular associates between βA 6–34 and BSB peptides (Soto's LPFFD, Tjernberg's KLVFF and two new ones) were calculated theoretically by AMBER force field based docking algorithms. The calculated structures emphasize the directing effect and pivotal role of electrostatic forces and the importance of the hydrophobic interactions of the side-chains in binding of BSB peptides to the β-amyloid peptide.

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Crystal structure of human GM2-activator protein with a novel β-cup topology

GM2 activator protein (GM2-AP) belongs to a small group of non- enzymatic lysosomal proteins that act as cofactors in the sequential degradation of gangliosides. It has been postulated that GM2-AP extracts single GM2 molecules from membranes and presents them in soluble form to β-hexosaminidase A for cleavage of N-acetyl- d-galactosamine and conversion to GM3. The high affinity of GM2-AP for GM2 is based on specfic recognition of the oligosaccharide moiety as well as the ceramide lipid tail. Genetic defects in GM2-AP result in an atypical form of Tay-Sachs disease known as variant AB GM2 gangliosidosis. The 2.0 Å resolution crystal structure of GM2-AP reported here reveals a previously unobserved fold whose main feature is an eight-stranded cup-shaped anti-parallel β-pleated sheet. The striking feature of the GM2-AP structure is that it possesses an accessible central hydrophobic cavity rather than a buried hydrophobic core. The dimensions of this cavity (12 Å × 14 Å × 22 Å) are suitable for binding 18-carbon lipid acyl chains. Flexible surface loops and a short α-helix decorate the mouth of the β-cup and may control lipid entry to the cavity.

Secondary Structure of Chorion Proteins of the Teleostean Fish Dentex dentex by ATR FT-IR and FT-Raman Spectroscopy

FT-Raman spectroscopy and ATR-IR spectroscopy were applied to study the secondary structure of the eggshell (chorion) proteins of the teleostean fish Dentex dentex. Raman and IR spectra clearly indicate an abundance of antiparallel β-pleated sheet conformation in chorion proteins. This findingis further supported by analysis of the vibrational data by regression techniques and deconvolution procedures. Thus, the common morphological characteristics of D. dentex, Salmo gairdneri, and other teleostean fish chorions may be explained on the basis of common secondary structure features of their constituent proteins. A detailed understanding of the interactions that dictate the self-assembly of fish chorion proteins to form the fish eggshell awaits determination of amino acid sequences.

Molecular Dynamics Simulation of the Cyclic Decapeptide Antibiotic, Gramicidin S, in Dimethyl Sulfoxide Solution

We report on a 5 ns molecular dynamics simulation of the decapeptide antibiotic, gramicidin S (cyclo-(Leu-DPhe-Pro-Val-Orn)2) in dimethyl sulfoxide solution. Throughout the simulation the backbone ring maintains an antiparallel β-pleated sheet conformation with two type II‘ β-turns. The backbone dihedrals are relatively inflexible and do not undergo any conformational transitions. The simulation reproduces the Φ and Ψ angles derived from nuclear magnetic resonance spectroscopy to within 18° standard deviation on average. Correlations between the backbone dihedral angles are found. The side chain dihedrals are much more flexible, and the multiple conformational states of these are classified using a clustering technique. Side chain hydrogen-bonding involving the ornithine and phenylalanine residues is analyzed and discussed in the context of previous work.

Self-Assembled β-Sheet Architectures for Bone-Tissue Engineering

AbstractAdvances in the understanding of biomineralization processes in a variety of organisms have revealed the critical role of three-dimensional scaffolding architectures to create a highly functionalized surface. These complex matrices function on a variety of length scales ranging from the macromolecular (10–100 nm) to the cellular (1–10mm) and larger. One dominant structural motif found in many of these architectures is macromolecules containing antiparallel β-pleated sheets. These “hints” from Nature have lead to the iterative design and development of a novel multipurpose platform technology based on a self-assembled periodic peptide architecture for use in bone-tissue engineering. Combining molecular modeling, structural biochemistry and synthetic techniques, we have produced a β-sheet hollow tube peptide nanoassembly. Such a synthetic approach allows for the template's designed parameters of electrostatic, geometric and stereochemical complimentarily to match those of the desired biomineral. Consequently, these templates readily nucleate calcite. Future studies will investigate the in vitro osteoconductive and osteogenic properties of these templates.

Packing motifs as predictors of the propensity of antibody fragments to crystallize

A recurring theme in the crystallization of antibody fragments in our laboratory has been a packing pattern involving formation of intermolecular, antiparallel β-pleated sheets across two-fold axes. The most common motif is the antiparallel stacking of constant (C) domains of light (L) chain dimers or Fab molecules. Here, cross-molecule six-stranded sheets are produced by hydrogen-bonding interactions of three-residue polypeptide segments (triads), in the i, i+2 and i+4 positions of the final strands (designated 3–3) of the three-chain layers from two adjacent molecules. In the variable (V) domains the triads are supplied by the first strands (4–1) of the four-chain layers and the resulting cross-molecule sheets contain eight strands. The latter type of packing is more likely to be seen in crystals of Fv fragments (V domains only) than in those of L chain dimers or Fabs. Amongst the triads from either the V or C domains, there are on average four sets of backbone carbonyl and amide groups within hydrogen bonding distance (<3.2 Å) of each other. In at least one example, the adjacent antiparallel strands are sterically aligned, but only two of the appropriate sets of atoms are sufficiently close to meet the distance criteria for intermolecular hydrogen bonding. These observations have been used to construct a list of rules for predicting which types of L chain dimers, Fab and Fvs are likely to crystallize in these packing patterns.

Crystal structure of CcdB, a topoisomerase poison from E. coli

The crystal structure of CcdB, a protein that poisons Escherichia coli gyrase, was determined in three crystal forms. The protein consists of a five-stranded antiparallel β-pleated sheet followed by a C-terminal α-helix. In one of the loops of the sheet, a second small three-stranded antiparallel β-sheet is inserted that sticks out of the molecule as a wing. This wing contains the LysC proteolytic cleavage site that is protected by CcdA and, therefore, forms a likely CcdA recognition site. A dimer is formed by sheet extension and by extensive hydrophobic contacts involving three of the five methionine residues and the C terminus of the α-helix. The surface of the dimer on the side of the α-helix is overall negatively charged, while the opposite side as well as the wing sheet is dominated by positive charges. We propose that the CcdB dimer binds into the central hole of the 59 kDa N-terminal fragment of GyrA, after disruption of the head dimer interface of GyrA.

Laser-Raman and FT-IR spectroscopic studies of peptide-analogues of silkmoth chorion protein segments

Silkmoth chorion, the proteinaceous major component of the eggshell, with extraordinary mechanical and physiological properties, consists of a complex set of proteins, which have a tripartite structure: a central, evolutionarily conserved, domain and two more variable `arms'. Peptide-analogues of silkmoth chorion protein central domain segments have been synthesized. Laser-Raman and infrared spectroscopic studies suggest the preponderance of antiparallel β-pleated sheet structure for these peptides, both in solution and in the solid state.

Progress in programming antibody fragments to crystallize

Completion of the X-ray analysis of the human B7-15A2 Fab opened a new vista (Immunotechnology 3, no. 4). In the crystal lattice, both the λ-type light chain (CL domain) and γ1-type heavy chain (CH1 domain) participated in formation of antiparallel β-pleated sheets with neighboring molecules related to the reference Fab by 2-fold axes. This observation evoked memories of the first description of this type of packing for human Bence-Jones (λ chain) dimers 20 years ago (Ely K.R. et al. Biochemistry 1978;17:158–167). Reexamination of packing interactions in selected crystal systems revealed that the C domains of λ and γ1 chains were structurally amenable to the formation of such cross-molecule β-structures, but κ chain CL domains were not. In the latter, a single proline residue disrupted the order of β-strand 3–3 in the middle of the surface used in λ and γ1 chains for intermolecular interactions with symmetry-related molecules. For the packing of Fv molecules, the VL domains are structurally well suited for analogous packing interactions through antiparallel 4–1 β-strands in adjacent molecules. Such interactions have been shown to provide the driving force in the crystal packing of a human (Pot) Fv from an IgM-κ cryoglobulin. Together, these observations suggest several avenues through which propensity to crystallize can be programmed into the designs of synthetic human Fabs, Fvs and single-chain antibodies.

Spectroscopic studies of Manduca sexta and Sesamia nonagrioides chorion protein structure

The secondary structure of Manduca sexta and Sesamia nonagrioides chorion proteins has been studied in intact chorions using laser-Raman and Fourier transform infra-red (FTIR) spectroscopy and in a solution containing extracted and reassembled chorion proteins using circular dichroism (CD) spectroscopy. Laser-Raman and IR spectra suggest the predominance of antiparallel β-pleated sheet structure in intact chorion proteins of both Lepidoptera species. The bands at 1673, 1674 cm −1 (amide I) and 1234–1238 cm −1 (amide III) in the laser-Raman spectra can best be interpreted as resulting from abundant antiparallel β-pleated sheet structure. Analysis of the amide I band suggests that chorion proteins consist of 60–70% antiparallel β-pleated sheet and 30–40% β-turns. Supporting evidence for the prevalence of antiparallel β-pleated sheet in chorion proteins was supplied using FTIR spectroscopy by the observation of a very intense absorption band at 1635 cm −1(amide I) and of a weak band at 1530, 1525 cm −1 (amide II) from chorions of both species. Surprisingly, analysis of the CD spectra of extracted and reassembled chorion proteins suggests that, in solution, they retain a regular secondary structure most probably dominated by β-pleated sheet. We therefore suggest that the prominent regular β-sheet structure of chorion proteins may exist in solution and dictate the aggregation and polymerization process in vivo.

Thioflavine T interaction with amyloid β-sheet structures

Thioflavine T (ThT) interacts in tissue sections with amyloid deposits comprised of a variety of protein species giving a characteristic fluorescent complex. Binding of the dye to amyloid fibrils in suspension generates an amyloid-specific fluorescent signal. This interaction with amyloid fibrils formed from different polypeptides and proteins containing antiparallel β-pleated sheet secondary structure is selective, occurring strongly with amyloid fibrils formed from insulin, transthyretin, polyglycine(I), Aβ (1–40), and weakly with β2–microglobulin. No fluorescence changes were seen with β-sheet fibrillar poly-L-lysine, islet amyloid peptide (20–29), or poly-L-serine. Native forms of transthyretin, insulin, β2-microglobulin, poly-L-lysine, Aβ(1–40), or several proteins containing high percentages of β-sheet were also unreactive. The affinity of the amyloid fibrils for ThT varied: (apparent Kd's 0.033 – 10 μ Amyloid A protein <insulin <ApoAII <polyglycine <transthyretin <Aβ(1–40). The spectral changes ind...

Crystallization, structure determination and least-squares refinement to 1.75 Å resolution of the fatty-acid-binding protein isolated from Manduca sexta L

The molecular structure of an insect fatty-acid-binding protein isolated from Manduca sexta L. has been determined and refined to a nominal resolution of 1.75 Å. Crystals used in the investigation were grown from 1.6 m-ammonium sulfate solutions buffered at pH 4.5 with 50 m m-sodium succinate, and belonged to space group P2 1 with unit cell dimensions of a = 27.5 A ̊ , b = 71.0 A ̊ , c = 28.7 A ̊ and β = 90.8 ° . An electron density map, phased with four heavy-atom derivatives and calculated to 2.5 Å resolution, allowed for complete tracing of the 131 amino acid residue polypeptide chain. Subsequent least-squares refinement of the model reduced the R-factor from 46.0% to 17.3% using all measured X-ray data from 30.0 Å to 1.75 Å. Approximately 92% of the amino acid residues fall into classical secondary structural elements including ten strands of anti-parallel β-pleated sheet, two α-helices, one type I turn, three type II turns, four type II′ turns and one type III turn. As in other fatty-acid-binding proteins, the overall molecular architecture of the insect molecule consists of ten strands of anti-parallel β-pleated sheet forming two layers that are nearly orthogonal to one another. A helix-turn-helix motif at the N-terminal portion of the protein flanks one side of the up-and-down β-barrel. The functional group of the fatty acid is within hydrogen-bonding distance of Gln39, Tyr129, Arg127 and a sulfate molecule, while the aliphatic portion of the ligand is surrounded by hydrophobic amino acid residues lining the β-barrel. The binding of the carboxylic acid portion of the ligand is very similar to that observed in P2 myelin protein and the murine adipocyte lipid-binding protein, but the positioning of the hydrocarbon tail after approximately C6 is completely different.

X-ray structure determination of telokin, the C-terminal domain of myosin light chain kinase, at 2.8 Å resolution

The three-dimensional structure of telokin, an acidic protein identical to the C-terminal portion of smooth muscle myosin light chain kinase from turkey gizzard, has been determined at 2·8 Å resolution and refined to a crystallographic R-factor of 19·5% for all measured X-ray data from 30 Å to 2·8 Å. Crystals used in the investigation belonged to the space group P3 221, with one molecule per asymmetric unit and unit cell dimensions of a = b = 64·4 A ̊ and c = 50·6 A ̊ . Telokin contains 154 amino acid residues, 103 of which were visible in the electron density map. The overall molecular fold of telokin consists of seven strands of antiparallel β-pleated sheet that wrap around to form a barrel. There is also an extended tail of eight amino acid residues at the N terminus that does not participate in β-sheet formation. The β-barrel can be simply envisioned as two layers of β-sheet, nearly parallel to one another, with one layer containing four and the other three β-strands. This type of β-barrel, as seen in telokin, was first observed for the CH2 domain of an immunoglobulin fragment Fc. Telokin is an intracellular protein and, as such, does not contain the disulphide linkage between β-strands B and F normally observed in the immunoglobulin constant domains. It does, however, contain two cysteine amino acid residues (Cys63 and Cys115) that are situated at structurally identical positions to those forming the disulphide linkage in the immunoglobulin constant domain.

Pyrularia Thionin: Physical Properties, Biological Responses and Comparison to Other Thionins and Cardiotoxin

Pyrularia thionin (PT) is a unique thionin which occupies a position between the viscotoxins and Gramineae thionins. Like the viscotoxins, PT comes from a dicotyledonous plant, Pyrularia pubera, and PT has a higher degree of amino acid homology with the viscotoxins than it does with the cereal grain thionins (1). However, PT has 4 disulfide bonds, as do the cereal grain thionins, and the viscotoxins have only 3. PT is distinct from both in that it is monomorphic. All thionins interact with and increase the permeability of cell membranes and are cytotoxic to some degree. PT and cardiotoxins also depolarize cell membranes, and PT opens a Ca2+ channel. PT as well as the other thoinins are amphipathic. This is apparent in the reported three dimensional structure of α-1 purothionin, which clearly shows distinct hydrophilic and hydrophobic domains on the surface of the protein, which contains two helical and one anti-parallel β-pleated sheet region. Examination of the physical properties show that PT combines with acidic phospholipids in synthetic membranes, and shows some specificity for phosphatidylserine. Our thesis is that PT combines with a specific phospholipid domain on the cellular membrane, most likely contining phosphatidylserine, causing early changes in membrane properties, including an increase in order parameter of the phospholipids, an increase in membrane permeability, depolarization of the membrane and opening of a Ca2+ channel. Later membrane responses include blebbing of the membrane bilayer and activation of an endogenous phospholipase A2. The activation of this enzyme results in membrane degradation and is the major cause of the long term cellular responses attributed to the basic peptide, including killing of cells in culture and neurotoxicity to animals. The cause of PT-induced hemolytic activity is not immediately apparent. In its binding properties, physical responses of the membrane and biological responses of cells, cardiotoxin from cobra venom behaves in a very similar manner to PT, indicating a similar mechanism of action for both toxic peptides.

Tandemly repeating peptide motifs and their secondary structure in Ceratitis capitata eggshell proteins Ccs36 and Ccs38

Evidence from amino acid composition, Fourier transform analysis of primary structure and secondary structure prediction suggests a tripartite structure for Ceratitis capitata eggshell proteins Ccs36 and Ccs38, which consists of a central domain and two flanking ‘arms’. The proteins, apparently, contain tandemly repeating peptide motifs specific for each domain of the tripartite structure. The central domain of both proteins, which exhibits extensive sequence homology with the corresponding domains of Drosophila melanogaster proteins s36 and s38, is formed by tandem repeats of an octapeptide-X-X-X-Z-Z-Z-Z-Z- (where X= large hydrophobic residue and Z = β-turn former residue) and its variants. It is predicted to adopt a compact, most probably twisted, antiparallel β-pleated sheet structure of β-sheet strands regularly alternating with β-turns or loops. The central domains of Ccs36 and Ccs38 share structural similarities, but they are recognizably different. The ‘arms’ of the proteins presumably serving for protein and species-specific functions differ substantially from those of Drosophila melanogaster. In Ccs36, the C-terminal ‘arm’ is formed by, almost precise, tandem repeats of an octapeptide -Y-X-A-A-P-A-A-S- (X = G or S), whereas the N-terminal ‘arm’ contains repeats of the predicted, alternating with β-turns. In the Ccs38 C-terminal ‘arm’ nonapeptide repeats of the form -Y-Z-Z-Z-Z-(G,A)-Z-Q-Z-(Z = A, G, P or S) are observed, whereas the N-terminal includes repeats of an octapeptide motif -x-y-G-z-G-u-G-v- ( x = I, S, y = G, Q). The latter are predicted as β-sheet strands alternating with β-turns, whereas, for the former the evidence is conflicting. The presence of these motifs suggests periodical patterns of dityrosine crosslinks, which harden the eggshell rendering it insoluble. Fourier transform infrared spectroscopy data from intact Ceratitis capitata eggshells support the validity of prediction.

Aggregation and secondary structure of synthetic amyloid βA4 peptides of Alzheimer's disease

The deposition of amyloid βA4 in the brain is a major pathological hallmark of Alzheimer's disease. Amyloid βA4 is a peptide composed of 42 or 43 amino acid residues. In brain, it appears in the form of highly insoluble, filamentous aggregates. Using synthetic peptides corresponding to the natural βA4 sequence as well as analog peptides, we demonstrate requirements for filament formation in vitro. We also determine aggregational properties and the secondary structure of βA4. A comparison of amino-terminally truncated βA4 peptides identifies a peptide spanning residues 10 to 43 as a prototype for amyloid βA4. Infrared spectroscopy of βA4 peptides in the solid state shows that their secondary structure consists of a β-turn flanked by two strands of antiparallel β-pleated sheet. Analog peptides containing a disulfide bridge were designed to stabilize different putative β-turn positions. Limited proteolysis of these analogs allowed a localization of the central β-turn at residues 26 to 29 of the entire sequence. Purified βA4 peptides are soluble in water. Size-exclusion chromatography shows that they form dimers that, according to circular dichroism spectroscopy, adopt a β-sheet conformation. Upon addition of salts, the bulk fraction of peptides precipitates and adopts a β-sheet structure. Only a small fraction of peptides remains solubilized. They are monomeric and adopt a random coil conformation. This suggests that the formation of aggregates depends upon a hydrophobic effect that leads to intra- and intermolecular interactions between hydrophobic parts of the βA4 sequence. This model is sustained by the properties of βA4 analogs in which hydrophobic residues were substituted. These peptides show a markedly increased solubility in salt solutions and have lost the ability to form filaments. In contrast, the substitution of hydrophilic residues leads only to small deviations in the shape of filaments, indicating that hydrophilic residues contribute to the specificity of interactions between βA4 peptides.

The structure of tumour necrosis factor - implications for biological function

The three-dimensional structure of TNF has been determined at 0.29 nm using the technique of X-ray crystallography. Published data on site-directed mutagenesis and antibody binding may now be assessed in the light of the structure, thus the links between structure and function for TNF may be addressed. TNF is a compact trimer composed of three identical subunits of 157 amino acids. The main-chain topology for a single subunit is essentially a beta-sandwich structure formed by two anti-parallel beta-pleated sheets. This mainchain fold corresponds to the 'jelly roll' motif observed in viral coat proteins such as VP1, VP2 and VP3 of rhinovirus, or the hemagglutinin molecule of influenza. TNF is the first non-viral protein to contain this motif. The subunits associate tightly about a threefold axis interacting through a simple edge-to-face packing of the beta-sandwich to form the solid, conical shaped trimer. A large number of the residues conserved between the amino acid sequences of TNF and lymphotoxin lie within the beta-sandwich or at the threefold axis of the trimer. This implies the presence of the same beta-sandwich motif in the lymphotoxin monomer and preservation of the edge-to-face mode of trimeric association. The detailed three dimensional structure for TNF explains a wide range of observations, including data on antibody binding and site directed mutagenesis. The currently available evidence points to a region of biological importance situated at the interface between two subunits on the lower half of the trimer.

1H-nuclear magnetic resonance studies of the neuropeptide head activator

The 1H-NMR spectrum of the neuropeptide head activator in aqueous solution has been completely assigned by two-dimensional NMR spectroscopy and selective deuteration. The apparent pseudo-first-order exchange rate, k ex, of the backbone amide protons and the correspondent activation enthalpies, Δ H ‡, were determined. The exchange rates decrease and the activation enthalpies increase from the N-terminal to the C-terminal part of the peptide. The exchange rates vary from 21 to 0.3 s −1 at 274 K, the activation enthalpies from 60 to 75 kJ · mol −1. The p K values of the terminal car☐yl group and of the lysine amino group have been estimated as 3.3 and 10.3, respectively. The NMR results are in line with a dimeric structure in an antisymmetric arrangement of the subunits, forming an antiparallel β-pleated sheet between C-terminal segments. The peptide bonds between pGlu-1, Pro-2 and Pro-3 are predominantly in trans-configuration, in fact no cis-isomers can be observed spectroscopically. The structure appears to be very stable; in the temperature and pH range studied, i.e., from 274 to 338 K and from pH 0.8 to pH 11.6, there are no spectroscopic indications for a global structural change.