Antiparallel Beta-sheet Structure Research Articles

Unveiling a New Antimicrobial Peptide with Efficacy against P. aeruginosa and K. pneumoniae from Mangrove-Derived Paenibacillus thiaminolyticus NNS5-6 and Genomic Analysis.

This study focused on the discovery of the antimicrobial peptide (AMP) derived from mangrove bacteria. The most promising isolate, NNS5-6, showed the closest taxonomic relation to Paenibacillus thiaminolyticus, with the highest similarity of 74.9%. The AMP produced by Paenibacillus thiaminolyticus NNS5-6 exhibited antibacterial activity against various Gram-negative pathogens, especially Pseudomonas aeruginosa and Klebsiella pneumoniae. The peptide sequence consisted of 13 amino acids and was elucidated as Val-Lys-Gly-Asp-Gly-Gly-Pro-Gly-Thr-Val-Tyr-Thr-Met. The AMP mainly exhibited random coil and antiparallel beta-sheet structures. The stability study indicated that this AMP was tolerant of various conditions, including proteolytic enzymes, pH (1.2-14), surfactants, and temperatures up to 40 °C for 12 h. The AMP demonstrated 4 µg/mL of MIC and 4-8 µg/mL of MBC against both pathogens. Time-kill kinetics showed that the AMP acted in a time- and concentration-dependent manner. A cell permeability assay and scanning electron microscopy revealed that the AMP exerted the mode of action by disrupting bacterial membranes. Additionally, nineteen biosynthetic gene clusters of secondary metabolites were identified in the genome. NNS5-6 was susceptible to various commonly used antibiotics supporting the primary safety requirement. The findings of this research could pave the way for new therapeutic approaches in combating antibiotic-resistant pathogens.

Site-Specific Structural Changes in Unmodified and Pyroglutamylated Amyloid Beta Peptide by Isotope-Edited Ftir

Amyloid beta-peptide (Abeta) forms cytotoxic assemblies that contribute to Alzheimer's disease. Recent evidence indicates that prefibrillar aggregates and not the fibrillar deposits exert the main toxic effect. In addition, naturally occurring N-terminally truncated and pyroglutamylated peptide (pE-Abeta) displays augmented cytotoxicity by an unknown mechanism. This study examines the conformational changes in both unmodified Abeta and pE-Abeta upon exposure to an aqueous environment. FTIR and circular dichroism were used to identify alpha-helix-to-beta-sheet conformational transitions of both peptides during aggregation. To gain site-specific structural information, the peptides were 13C,15N-labeled at residues 16-18 (KLV) or 36-39 (VGGV), followed by FTIR analysis. The peptides dried from hexafluoroisopropanol were alpha-helical (amide I peak at 1660-1657 cm-1) and showed negligible intensity of the labeled segments around 1615 cm-1, suggesting that in alpha-helical conformation the labeled amide groups behave like isolated oscillators. Upon addition of aqueous buffer (pH 7.2) both peptides rapidly adopted beta-sheet structure (amide I peak at 1637-1629 cm-1), with disproportionally prominent components around 1604-1597 cm-1 generated by the labeled segments. The intensity and the frequency of the amide I mode of the isotope-labeled segments suggest 12C-13C vibrational coupling, consistent with formation of antiparallel beta-sheet structures. Moreover, the amide I contours of the peptides under near-physiological and low ionic strength conditions were significantly different; both peptides exhibited an increased alpha-helical and decreased beta-sheet propensity under low ionic strength conditions, indicating a strong influence of the ionic strength on the aggregation kinetics and accompanying structural changes. Ongoing studies focus on structural differences between the unmodified Abeta and pE-Abeta peptides as well as their mutual structural effects when combined at various molar ratios, in an attempt to understand the structural basis of the elevated cytotoxicity of pE-Abeta.

Molecular Mechanisms of Misfolding of Amyloid Peptides

The current model for the development of Alzheimer's, Parkinson's, Huntington's, prion, and other neurodegenerative diseases involves protein misfolding as the early step followed by spontaneous aggregation, with specific proteins identified as the primary initiators for disease development. Therefore, elucidating the properties of the disease-prone misfolded states, understanding the mechanism of their formation, and identification of their most toxic forms will open prospects for the development of early diagnostics and specific therapeutics for these diseases. We have developed single molecule AFM force spectroscopy (SMFS) experimental approach enabling us to probe interprotein interactions and to identify those interactions that correspond to misfolded protein states. Using SMFS, we have discovered that the misfolded dimers are very stable and have a lifetime in a second time scale. Such a long lifetime of dimers suggests that the formation of dimers is the mechanism by which the protein misfolded state is stabilized. We hypothesize that the formation of highly stable misfolded dimers is a critical step in the entire process of the peptide self-assembly into aggregates. The Molecular Dynamics (MD) simulation performed at the μs timescale demonstrated that isolated non-structured monomer upon approaching to each other changed dramatically their initial conformation and formed dimers with antiparallel beta-sheet structures. Steered MD approach showed that the dimers dissociated cooperatively resulting in a sharp rupture peak corresponding to breakage of the beta-sheet structure. Altogether, the SMFS experimental study and computational analysis revealed a critical role of the interpetide interaction in the misfolding process and highlighting the key role of the dimerization in the amyloid aggregation process. Acknowledgements: Grants from NIH (5R01 GM096039-02) and NSF (EPS-1004094).

The roles of macromolecules in imatinib resistance of chronic myeloid leukemia cells by Fourier transform infrared spectroscopy

Imatinib is a first generation tyrosine kinase inhibitor, which is used for the treatment of chronic myeloid leukemia. However, resistance to imatinib is an important problem. Different mechanisms have been explained for imatinib resistance. In this study, we examined the roles of macromolecules in imatinib resistance in K562 cells at the molecular level using Fourier Transform Infrared (FT-IR) spectroscopy. An amount of 3μM imatinib resistant cells were generated by our group and named as K562/IMA-3 cells. Changes in macromolecules in parental and resistant cells were studied by FT-IR spectroscopy. Imatinib resistance caused changes, which indicated decreases in the level of glycogen and increases in the membrane order. The amount of unsaturated lipids increased in the imatinib resistant cells indicating lipid peroxidation. Imatinib resistance caused changes in the lipid/protein ratio. The relative protein content increased with respect to nucleic acids indicating higher transcription and protein expression and structural/organizational changes in the nucleus were evident as revealed by frequency changes in the nucleic acid bands. Changes in the amide bands revealed changes in the proteome of the resistant cells. Protein secondary structural changes indicated that the antiparallel beta sheet's structure increased, however the alpha helix structure, beta sheet structure, random coil structure and turns decreased in the resistant cells. These results indicate that the FT-IR technique provides a suitable method for analyzing drug resistance related structural changes in leukemia and other cancer types.

Macromolecular Changes in Nilotinib Resistant K562 Cells; an In vitro Study by Fourier Transform Infrared Spectroscopy

Nilotinib is a second generation tyrosine kinase inhibitor which is used in both first and second line treatment of chronic myeloid leukemia (CML). In the present work, the effects of nilotinib resistance on K562 cells were investigated at the molecular level using Fourier transform infrared (FT-IR) spectroscopy. Human K562 CML cells were exposed to step-wise increasing concentrations of nilotinib, and sub-clones of K562 cells resistant to 50nM nilotinib were generated and referred to as K562/NIL-50 cells. Antiproliferative effects of nilotinib were determined by XTT cell proliferation assay. Changes in macromolecules in parental and resistant cells were studied by FT-IR spectroscopy. Nilotinib resistance caused significant changes which indicated increases in the level of glycogen and membrane/lipid order. The amount of unsaturated lipids increased in the nilotinib resistant cells indicating lipid peroxidation. The total amount of lipids did not change significantly but the relative proportion of cholesterol and triglycerides altered considerably. Moreover, the transcriptional status decreased but metabolic turn-over increased as revealed by the FT-IR spectra. In addition, changes in the proteome and structural changes in both proteins and the nucleus were observed in the K562/NIL-50 cells. Protein secondary structural analyses revealed that alpha helix structure and random coil structure decreased, however, anti-parallel beta sheet structure, beta sheet structure and turns structure increased. These results indicate that the FT-IR technique provides a method for analyzing drug resistance related structural changes in leukemia and other cancer types.

Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli

BackgroundIn recent years, it has been gradually realized that bacterial inclusion bodies (IBs) could be biologically active. In particular, several proteins including green fluorescent protein, β-galactosidase, β-lactamase, alkaline phosphatase, D-amino acid oxidase, polyphosphate kinase 3, maltodextrin phosphorylase, and sialic acid aldolase have been successfully produced as active IBs when fused to an appropriate partner such as the foot-and-mouth disease virus capsid protein VP1, or the human β-amyloid peptide Aβ42(F19D). As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further.ResultsIn this paper, we report that an ionic self-assembling peptide ELK16 (LELELKLK)2 was able to effectively induce the formation of cytoplasmic inclusion bodies in Escherichia coli (E. coli) when attached to the carboxyl termini of four model proteins including lipase A, amadoriase II, β-xylosidase, and green fluorescent protein. These aggregates had a general appearance similar to the usually reported cytoplasmic inclusion bodies (IBs) under transmission electron microscopy or fluorescence confocal microscopy. Except for lipase A-ELK16 fusion, the three other fusion protein aggregates retained comparable specific activities with the native counterparts. Conformational analyses by Fourier transform infrared spectroscopy revealed the existence of newly formed antiparallel beta-sheet structures in these ELK16 peptide-induced inclusion bodies, which is consistent with the reported assembly of the ELK16 peptide.ConclusionsThis has been the first report where a terminally attached self-assembling β peptide ELK16 can promote the formation of active inclusion bodies or active protein aggregates in E. coli. It has the potential to render E. coli and other recombinant hosts more efficient as microbial cell factories for protein production. Our observation might also provide hints for protein aggregation-related diseases.

Genomic Evidence for Elements of a Programmed Cell Death Pathway In Plasmodium: Exploiting Programmed Parasite Death for Malaria Control?

AbstractAbstract 4226Malaria, caused by species of the Plasmodium parasite, remains a major global health burden. To combat the disease, new areas of investigation are constantly developed with a view to understanding the biology of the organism and identifying potential drug targets. A new line of enquiry has emerged concerning the occurrence of parasite programmed cell death (PCD); however, there are conflicting experimental reports regarding the presence and phenotype of PCD in Plasmodium species. Furthermore, very little genomic evidence exists for a PCD pathway in this genus with only caspase-like domains being detected. One reason for the limited availability of genomic data may be the unusual characteristics of the P. falciparum genome, such as the extreme AT bias. In this study we examined the complete genome sequences of four Plasmodium species (P. falciparum, P. vivax, P. yoelii and P. knowlesi) for evidence of a p53-dependant PCD pathway. This pathway is well characterized in animals, and elements of the molecular machinery have been identified in protozoa. In addition, p53-like responses leading to apoptosis (a common PCD phenotype) have been demonstrated in phylogenetically diverse eukaryotes, including animals, protozoa, green algae and plants, suggesting that the origin of p53-initiated PCD is evolutionary ancient leading us to believe that a similar genomic toolkit may exist in Plasmodium. A detailed analysis of the four Plasmodium genomes was performed using an array of computational approaches, which included standard homology methods, phylogenetics, structural models and a novel evolutionary rate-based alignment algorithm FIRE (Functional Inference using the Rates of Evolution), which was developed to identify homologous and analogous genes in organisms with unusual genomes, such as P. falciparum, and hence low sequence similarity. Homology methods uncovered key elements of a classical PCD pathway, including ATM, MDM2, CR6 and three peptidase C14 (catalytic caspase) domains in each of the four Plasmodium genomes. Phylogenetic analysis demonstrated that the peptidase C14 (caspase) domains are evolutionary ancient and cluster with other PCD-related caspases suggesting that they are involved in a PCD pathway as opposed to other cellular functions that may use similar domains. In addition, highly sensitive hidden Markov models retrieved 15 sequences with low similarity to the eukaryotic p53 DNA-binding domain (DBD). Further analysis revealed that two P. falciparum sequences (accession numbers PFE1120w and PF11_0091 in the Plasmodium database www.PlasmoDB.org version 6.5) may be p53 DBD-like sequences. Both have similar evolutionary rates across codons, as well as anti-parallel beta sheet structures with Greek key topology, which are a characteristic feature of known p53 DBDs. Whether either of these proteins has functional significance for the PCD pathway in P. falciparum requires empirical verification. However, these data and the existence of p53-like activity in plants and algae, suggest that this pathway has an ancient origin and provide the first evidence for a p53-dependant PCD pathway in malaria parasites. The presence of this genomic toolkit raises questions regarding the potential role of this pathway in the P. falciparum lifecycle. The phenomenon of programmed parasite death may have adaptive significance and proteins in the PCD pathway may potentially be sufficiently different from the human orthologs, given the low level of sequence similarity. These findings could thus open a new line of investigation for novel malaria drug targets. Disclosures:No relevant conflicts of interest to declare.

Interruptions in the collagen repeating tripeptide pattern can promote supramolecular association

The standard collagen triple-helix requires a perfect (Gly-Xaa-Yaa)(n) sequence, yet all nonfibrillar collagens contain interruptions in this tripeptide repeating pattern. Defining the structural consequences of disruptions in the sequence pattern may shed light on the biological role of sequence interruptions, which have been suggested to play a role in molecular flexibility, collagen degradation, and ligand binding. Previous studies on model peptides with 1- and 4-residue interruptions showed a localized perturbation within the triple-helix, and this work is extended to introduce natural collagen interruptions up to nine residue in length within a fixed (Gly-Pro-Hyp)(n) peptide context. All peptides in this set show decreases in triple-helix content and stability, with greater conformational perturbations for the interruptions longer than five residue. The most stable and least perturbed structure is seen for the 5-residue interruption peptide, whose sequence corresponds to a Gly to Ala missense mutation, such as those leading to collagen genetic diseases. The triple-helix peptides containing 8- and 9-residue interruptions exhibit a strong propensity for self-association to fibrous structures. In addition, a small peptide modeling only the 9-residue sequence within the interruption aggregates to form amyloid-like fibrils with antiparallel beta-sheet structure. The 8- and 9-residue interruption sequences studied here are predicted to have significant cross-beta aggregation potential, and a similar propensity is reported for approximately 10% of other naturally occurring interruptions. The presence of amyloidogenic sequences within or between triple-helix domains may play a role in molecular association to normal tissue structures and could participate in observed interactions between collagen and amyloid.

Functional analysis of the activation domain of RF2a, a rice transcription factor

Rice transcription factor RF2a binds to the BoxII cis element of the promoter of rice tungro bacilliform virus and activates promoter expression. The acidic acid-rich domain of RF2a is a transcription activator and has been partially characterized (Dai et al., 2003). The RF2a acidic domain (A; amino acids 49-116) was fused with the synthetic zinc finger ZF-TF 2C7 and was co-introduced with a reporter gene into transgenic Arabidopsis plants. Expression of the reporter gene was increased up to seven times by the effector. In transient assays in tobacco BY-2 protoplasts, we identified a subdomain comprising amino acids 56-84 (A5) that was equally as effective as an activator as the entire acidic domain. A chemically inducible system was used to show determined that A and A5 domains are equally as effective in transcription activation as the well-characterized VP16 activation domain. Bioinformatics analyses revealed that the A5 domain is present only in b-ZIP transcription factors. In dicots, the A domain contains an insertion of four amino acids that is not present in monocot proteins. The A5 domain, and similar domains in other b-ZIP transcription factors, is predicted to form an anti-parallel beta sheet structure.

Tetrapeptide-Based Hydrogels: for Encapsulation and Slow Release of an Anticancer Drug at Physiological pH

Here, we report two synthetic oligopeptide-based, thermoreversible, pH-sensitive hydrogels. In gel phase, these self-assembling tetrapeptides form a long interconnected nanofibrilar network structure, as is evident from various microscopic techniques, including field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). FTIR, circular dichroism, and wide angle X-ray diffraction (WAXD) favor an antiparallel beta-sheet structure of these gelators in the gel state. Finally, these hydrogels have been utilized for entrapment and slow release of an anticancer drug doxorubicin at physiological pH, promising their future application as a drug delivery vehicle.

Nanofibers from Oxazolidi‐2‐one Containing Hybrid Foldamers: What is the Right Molecular Size?

A series of oligomers of the type Boc-(L-Phe-D-Oxd)(n)-OBn (Boc = tert-butoxycarbonyl; Oxd = 4-methyl-5-carboxy oxazolidin-2-one; Bn = benzyl) were prepared for n = 2-5. The shortest oligomer, Boc-(L-Phe-D-Oxd)(2)-OBn, aggregates and forms a fiber-like material with an anti-parallel beta-sheet structure in which the oligopeptide units are connected to each other by only one intermolecular hydrogen bond. The longer oligomers exhibit structural heterogeneity. They start to organize into secondary structures by the formation of intramolecular hydrogen bonds at the pentamer level. Microscopy and diffraction of the oligomers indicated a crystalline character for only the shorter ones.

Evidence for novel beta-sheet structures in Iowa mutant beta-amyloid fibrils.

Asp23-to-Asn mutation within the coding sequence of beta-amyloid, called the Iowa mutation, is associated with early onset, familial Alzheimer's disease and cerebral amyloid angiopathy, in which patients develop neuritic plaques and massive vascular deposition predominantly of the mutant peptide. We examined the mutant peptide, D23N-Abeta40, by electron microscopy, X-ray diffraction, and solid-state NMR spectroscopy. D23N-Abeta40 forms fibrils considerably faster than the wild-type peptide (k = 3.77 x 10(-3) min(-1) and 1.07 x 10(-4) min(-1) for D23N-Abeta40 and the wild-type peptide WT-Abeta40, respectively) and without a lag phase. Electron microscopy shows that D23N-Abeta40 forms fibrils with multiple morphologies. X-ray fiber diffraction shows a cross-beta pattern, with a sharp reflection at 4.7 A and a broad reflection at 9.4 A, which is notably smaller than the value for WT-Abeta40 fibrils (10.4 A). Solid-state NMR measurements indicate molecular level polymorphism of the fibrils, with only a minority of D23N-Abeta40 fibrils containing the in-register, parallel beta-sheet structure commonly found in WT-Abeta40 fibrils and most other amyloid fibrils. Antiparallel beta-sheet structures in the majority of fibrils are indicated by measurements of intermolecular distances through (13)C-(13)C and (15)N-(13)C dipole-dipole couplings. An intriguing possibility exists that there is a relationship between the aberrant structure of D23N-Abeta40 fibrils and the unusual vasculotropic clinical picture in these patients.

Macrocyclic Design Strategies for Small, Stable Parallel β-Sheet Scaffolds

Pairs of short peptide strands can be induced to adopt an antiparallel beta-sheet secondary structure in aqueous solution via a macrocyclic constraint, as illustrated by many natural and designed peptides. We show that an analogous strategy is successful for creation of small units of parallel beta-sheet secondary structure in aqueous solution. Cyclization in this case requires nonpeptide segments for N-to-N and C-to-C interstrand linkage. Surprisingly, we find that only one of these segments needs to be preorganized.

Structural, thermodynamic, and kinetic properties of Gramicidin analogue GS6 studied by molecular dynamics simulations and statistical mechanics

Gramicidin S (GS) analogues belong to an important class of cyclic peptides, characterized by an antiparallel double-stranded beta-sheet structure with Type II' beta-turns. Such compounds can be used as model systems to understand the folding/unfolding process of beta-hairpins and more in general of beta-structures. In the present study, we specifically investigate the folding/unfolding behavior of the hexameric Gramicidin S analogue GS6 by using all-atoms molecular dynamics (MD) simulations at different temperatures, coupled to a statistical mechanical model based on the Quasi Gaussian Entropy theory. Such an approach permits to describe the structural, thermodynamic, and kinetic properties of the peptide and to quantitatively characterize its folding/unfolding transitions.

Energy Landscapes Associated with the Self-Aggregation of an Alanine-Based Oligopeptide (AAKA)4

The alanine-based 16-mer peptide (AAKA)(4) has recently been shown to aggregate into a hydrogel at centimolar concentrations and high ionic strength ( Measey, T. J.; Schweitzer-Stenner, R. J. Am. Chem. Soc. 2006 , 128 , 13324. ). This is a surprising result since the current understanding of peptide self-aggregation would lead one to expect that the positively charged lysine residues inhibit the formation of beta-sheet structures. The present study was aimed at shedding some light on the mechanism governing the initial phase of the self-aggregation process. To this end we measured CD and FTIR spectra of the (AAKA)(n) at millimolar concentration and low ionic strength and found that the peptide forms soluble aggregates rather than a hydrogel under these conditions. To analyze the initial phase of this aggregation process, we carried out simulations on the dimerization and trimerization of (AAKA)(4) using replica exchange molecular dynamics (MD) methods based on an implicit solvent model. The results indicate that the peptide aggregates into stable antiparallel beta-sheet structures under conditions comparable to experiments. Furthermore, the existence of trimers is a very sensitive function of the peptide concentration and temperature; no significant amount of trimers is formed at low concentration and the trimer population drops sharply as temperature increases above about 320 K. These results on oligomers are consistent with the trend observed in our experiments (reported elsewhere). We have also obtained the free energy landscapes of the trimers as a function of reaction coordinates, which reveal a few basins of minimal free energy. Associated with them, six major types of stable conformers were identified and the structures of transition states between them were also determined. We suggest a possible transition mechanism between these conformers based on the landscape and structural analysis of the aggregates.

Structural Characterization of a Soluble Amyloid β-Peptide Oligomer

Alzheimer's disease (AD) is a neurodegenerative disorder that is linked to the presence of amyloid beta-peptides that can form insoluble fibrils or soluble oligomeric assemblies. Soluble forms are present in the brains and tissues of Alzheimer's patients, and their presence correlates with disease progression. Long-lived soluble forms can be generated in vitro by using small amounts of aliphatic hydrocarbon chains of detergents or fatty acids in preparations of amyloid beta-peptides. Using NMR, we have characterized soluble oligomers of Abeta preglobulomer and globulomer that are stable and alter synaptic activity. The NMR data indicate that these soluble forms have a mixed parallel and antiparallel beta-sheet structure that is different from fibrils which contain only parallel beta-sheets. Using the structural data, we engineered a disulfide bond into the soluble Abeta globulomer to give a "new" soluble antigen that is stable, homogeneous, and binds with the same affinity to selective antibodies as the parent wt globulomer.

Different Individual Amyloid Fibrils Exhibit Different Beta Sheet Secondary Structures via Near-field Infrared Spectroscopy

Morphology and secondary structure of individual amyloid fibrils synthesized from the #21-31 peptide fragment of B2-microglobulin were obtained using apertureless near-field scanning infrared microscopy (ANSIM). The sample exhibits a heterogeneous population of fibrils: some fibrils exhibit parallel and some with anti-parallel beta sheet structures. ANSIM is an optical technique which uses a carbon monoxide IR laser coupled with a tapping mode atomic force microscope using homodyne detection demodulated at the tip frequency. The experimental near-field spectra correlate strongly to the calculated near-field spectrum as well as the far-field spectrum. Near-field images exhibit high attenuation of the amyloid fibrils at approximately 1630 cm−1. Strong attenuation of the incident IR radiation at 1691 cm−1 corresponding to the presence of anti-parallel beta sheet structure is observed for a fraction of the fibrils. The observation of the anti-parallel beta sheet conformation is linked to the synthesis method used to produce these amyloid fibrils. It is shown that the addition of trimethylamine N-oxide (TMAO) can accelerate the formation of amyloid fibrils and induce anti-parallel beta sheet structure.

Conformation and hydrogen bonding of N-formylmethionyl peptides

Crystals of N-formyl-L-methionyl-L-phenylalanine (C15H20N2O4S), grown from aqueous methanol solution are orthorhombic, space group, P2(1)2(1)2(1), with cell parameters at 294K of a = 4.900(2), b = 17.947(4), c = 18.726(4)A, V = 1646.8A3, M.W. = 324.4, Z = 4 and Dm = 1.308 g/cc, and as expected, all nearly identical to that of N-f-D-Met-D-Phe studied by Jeffs, Heald, Chodosh & Eggleston (Int. J. Peptide Protein Res. 24, 442-446, 1984). The crystal structure was solved and refined using CAD-4 data (1095 reflections greater than or equal to 3 sigma) to a final R value of 0.042. Molecules related by the alpha-translation form a parallel beta-sheet rather than anti-parallel sheet as stated in the earlier study of Jeffs et al. The formation of the parallel rather than the anti-parallel beta-sheet structure, the use of the C-H ...O hydrogen bonds to stabilize the beta-sheet and the very short O-H ...O hydrogen bond between the carboxyl OH and the N-acyl oxygen atom emerge as the main structural features of the chemotactic N-formyl methionyl peptides.

A Novel Model System for Design of Biomaterials Based on Recombinant Analogs of Spider Silk Proteins

Spider dragline silk possesses impressive mechanical and biochemical properties. It is synthesized by a couple of major ampullate glands in spiders and comprises of two major structural proteins--spidroins 1 and 2. The relationship between structure and mechanical properties of spider silk is not well understood. Here, we modeled the complete process of the spider silk assembly using two new recombinant analogs of spidroins 1 and 2. The artificial genes sequence of the hydrophobic core regions of spidroin 1 and 2 have been designed using computer analysis of existing databases and mathematical modeling. Both proteins were expressed in Pichia pastoris and purified using a cation exchange chromatography. Despite the absence of hydrophilic N- and C-termini, both purified proteins spontaneously formed the nanofibrils and round micelles of about 1 microm in aqueous solutions. The electron microscopy study has revealed the helical structure of a nanofibril with a repeating motif of 40 nm. Using the electrospinning, the thin films with an antiparallel beta-sheet structure were produced. In summary, we were able to obtain artificial structures with characteristics that are perspective for further biomedical applications, such as producing three-dimensional matrices for tissue engineering and drug delivery.

Supramolecular Bidentate Ligands by Metal‐Directed in situ Formation of Antiparallel β‐Sheet Structures and Application in Asymmetric Catalysis

The principles of protein structure design, molecular recognition, and supramolecular and combinatorial chemistry have been applied to develop a convergent metal-ion-assisted self-assembly approach that is a very simple and effective method for the de novo design and the construction of topologically predetermined antiparallel beta-sheet structures and self-assembled catalysts. A new concept of in situ generation of bidentate P-ligands for transition-metal catalysis, in which two complementary, monodentate, peptide-based ligands are brought together by employing peptide secondary structure motif as constructing tool to direct the self-assembly process, is achieved through formation of stable beta-sheet motifs and subsequent control of selectivity. The supramolecular structures were studied by (1)H, (31)P, and (13)C NMR spectroscopy, ESI mass spectrometry, X-ray structure analysis, and theoretical calculations. Our initial catalysis results confirm the close relationship between the self-assembled sheet conformations and the catalytic activity of these metallopeptides in the asymmetric rhodium-catalyzed hydroformylation. Good catalyst activity and moderate enantioselectivity were observed for the selected combination of catalyst and substrate, but most importantly the concept of this new methodology was successfully proven. This work presents a perspective interface between protein design and supramolecular catalysis for the design of beta-sheet mimetics and screening of libraries of self-organizing supramolecular catalysts.