Controlled patterning of peptide nanotubes and nanospheres using inkjet printing technology.

Peptide nanostructures are expected to serve as a major tool in future nanotechnological applications owing to their excellent self-assembly properties, biological and chemical flexibility and structural simplicity. Yet one of the limiting factors for the integration of peptide assemblies into functional electro-organic hybrid devices is the controlled patterning of their assemblies. Here we report the use of inkjet technology for the application of peptide nanostructures on nonbiological surfaces. The aromatic dipeptides nanotubes (ADNT) which readily self-assemble in solution were used as an 'ink' and patterned on transparency foil and ITO plastic surfaces using a commercial inkjet printer. While inkjet technology was used in the past for the patterning of carbon nanotubes, it was not used for the deposition of biomolecular nanostructures. Furthermore, during the development of the application we were able to produce two types of nanostructures, i.e. nanotubes and nanospheres by the self-assembly of the same aromatic dipeptide, tertbutoxycarbonyl-Phe-Phe-OH (Boc-Phe-Phe-OH), under different conditions. Both spherical and tubular structures could be efficiently patterned on surfaces into predesigned patterns. The applications of such technology are discussed.     

Self-Assembly of Phenylalanine Oligopeptides: Insights from Experiments and Simulations

Studies of peptide-based nanostructures provide general insights into biomolecular self-assembly and can lead material engineering toward technological applications. The diphenylalanine peptide (FF) self-assembles into discrete, hollow, well ordered nanotubes, and its derivatives form nanoassemblies of various morphologies. Here we demonstrate for the first time, to our knowledge, the formation of planar nanostructures with β-sheet content by the triphenylalanine peptide (FFF). We characterize these structures using various microscopy and spectroscopy techniques. We also obtain insights into the interactions and structural properties of the FF and FFF nanostructures by 0.4-μs, implicit-solvent, replica-exchange, molecular-dynamics simulations of aqueous FF and FFF solutions. In the simulations the peptides form aggregates, which often contain open or ring-like peptide networks, as well as elementary and network-containing structures with β-sheet characteristics. The networks are stabilized by polar and nonpolar interactions, and by the surrounding aggregate. In particular, the charged termini of neighbor peptides are involved in hydrogen-bonding interactions and their aromatic side chains form “T-shaped” contacts, as in three-dimensional FF crystals. These interactions may assist the FF and FFF self-assembly at the early stage, and may also stabilize the mature nanostructures. The FFF peptides have higher network propensities and increased aggregate stabilities with respect to FF, which can be interpreted energetically.     

Designed aromatic homo-dipeptides: formation of ordered nanostructures and potential nanotechnological applications

Molecular self-assembly offers new routes for the fabrication of novel materials at the nano-scale. Peptide-based nanostructures represent nano-objects of particular interest, as they are biocompatible, can be easily synthesized in large amounts, can be decorated with functional elements and can be used in various biological and non-biological applications. We had previously revealed the formation of highly ordered tubular structures by the diphenylalanine peptide, the core recognition motif of Alzheimer's beta-amyloid polypeptide, due to specific aromatic interactions. We further confirmed this model and demonstrated that a non-charged peptide analogue, Ac-Phe-Phe-NH2, self-assembled into similar tubular structures. We later explored other amine and carboxyl modified diphenylalanine peptide analogues and revealed that these dipeptides can form ordered tubular structures at the nanometric scale. Moreover, a very similar peptide, the diphenylglycine, self-assembled into ordered nano-spherical assemblies. Here we extend our research and explore the self-assembly of other homo-aromatic dipeptides in which their phenyl side-chains are modified with halogen atoms (di-para-fluoro-Phe, di-pentafluoro-Phe, di-para-iodo-Phe), additional phenyl groups (di-4-phenyl-Phe), or with nitro substitutions (di-para-nitro-Phe). We also probed the effect of the alteration of the phenyl groups with naphtyl groups (di-D-1-Nal and di-D-2-Nal). In all cases, well-ordered nanostructures were obtained and studied by scanning electron microscopy, transmission electron microscopy and vibrational spectroscopy. Taken together, the current work and previous ones define the homo-aromatic dipeptide as a central motif for the formation of ordered self-assembled tubular, spherical and two-dimensional structures at the nano-scale.     

Acceleration of protein aggregation by amphiphilic peptides: transformation of supramolecular structure of the aggregates

Protein self-assembly and aggregation represent a special tool in biomedicine and biotechnology to produce biological materials for a wide range of applications. The protein aggregates are very different morphologically, varying from soluble amorphous aggregates to highly ordered amyloid-like fibrils, the latter being associated with molecular structures able to perform specific functions in living systems. Fabrication of novel biomaterials resembling natural protein assemblies has awakened interest in identification of low-molecular-weight biogenic agents as regulators of transformation of aggregation-prone proteins into fibrillar structures. Short amphiphilic peptides can be considered for this role. Using dynamic light scattering, turbidimetry, fluorescence spectroscopy, and transmission electron microscopy (TEM), we have demonstrated that the Arg-Phe dipeptide dramatically accelerates the aggregation of a model protein, α-lactalbumin, to generate morphologically different structures. TEM revealed transformation of spherical particles observed in the control samples into branched chains of fibril-like nanostructures in the presence of the peptide, suggesting that amphiphilic peptides can induce changes in the physicochemical properties of a protein substrate (net charge, hydrophobicity, and tendency to β-structure formation) resulting in accumulation of peptide-protein complexes competent to self-assembly into supramolecular structures. A number of other short amphiphilic peptides have also been shown to accelerate the aggregation process, using alternative complementary protein substrates for identification of molecular recognition modules. Peptide-protein assemblies are suggested to play the role of building blocks for formation of supramolecular structures profoundly differing from those of the individual protein substrate in type, size, and shape.     

Synthesis and mass spectrometric characterization of a metal-affinity decapeptide: copper-induced conformational changes

A decapeptide with high affinity toward heavy metal ions (RCHQYHHNRE) has been prepared by Fmoc strategy using TGR resin as solid support. The model peptide provides a simple system that can be used for a systematic study of the impact of different metal ions on peptide secondary structure on a molecular level; histidine residues were incorporated into the peptide in a sequence similar to beta-amyloid peptide (Abeta1-40) to generate possible complexation sites for Cu (2+) ions. The peptide secondary structure, as investigated by circular dichroism, and self-assembled nanostructures were observed to depend strongly on the presence of copper and sodium dodecyl sulfate (SDS). Atomic force microscopy (AFM) revealed also that copper and SDS affected slightly the Abeta1-40 nanostructures. An explanation for the effect of metal ions and SDS on the self-assembly of peptides was proposed. The extensive beta-sheet formation may further promote peptide self-assembly into longer fibers.     

Self-assembly of rationally designed peptides under two-dimensional confinement

The rational design of interfacially confined biomolecules offers a unique opportunity to explore the cooperative relationship among self-assembly, nucleation, and growth processes. This article highlights the role of electrostatics in the self-assembly of β-sheet-forming peptides at the air-water interface. We characterize the phase behavior of a periodically sequenced sheet-forming peptide by using Langmuir techniques, Brewster angle microscopy, attenuated total reflection Fourier transform infrared spectroscopy, and circular dichroism spectroscopy. We find that peptides with an alternating binary sequence transition at high pressures from discrete circular domains to fibrous domains. The qualitative behavior is independent of surface pressure but dependent on molecular areas. In addition, thermodynamic models are employed to specifically quantify differences in electrostatics by obtaining parameters for the critical aggregation area, the limiting molecular area, and the dimensionless ratio of line tension/dipole density. Using these parameters, we are able to relate localized charge distribution to phase transitions, which will allow us to apply these molecules to examine how the dynamics of self-assembly can be directly coupled to the formation of composite nanostructures in biology.     

Differential self-assembly behaviors of cyclic and linear peptides

Here we ask the fundamental questions about the effect of peptide topology on self-assembly. The study revealed that the self-assembling behaviors of cyclic and linear peptides are significantly different in several respects, in addition to sharing several similarities. Their clear differences included the morphological dissimilarities of the self-assembled nanostructures and their thermal stability. The similarities include their analogous critical aggregation concentration values and cytotoxicity profiles, which are in fact closely related. We believe that understanding topology-dependent self-assembly behavior of peptides is important for developing tailor-made self-assembled peptide nanostructures.     

Organic solvents mediate self-assembly of GAV-9 peptide on mica surface

Self-assembly of peptides into fibrils and other morphologies has attracted much attention inmany fields,especially in nanofabrication,pathology and biochemistry.In this paper,self-assembly of GAV-9peptide in organic solvents,ethanol and acetone,was investigated using atomic force microscopy(AFM)and attenuated total reflectance-Fourier transform infrared spectroscopy(ATR-FTIR).The results indicatedthat GAV-9 self-assembled into various nanostructures in both solvents after deposited and evaporated onmica.Fibrils with β-sheet conformation were observed in both solvents when the peptide concentrationwas higher than 280 μM.However,ordered fibrils with β-sheet conformation were formed in ethanol,butnot in acetone,with a peptide concentration ranging from 7 μM to 28 μM.We attribute the formation ofvarious nanostructures to the different physicochemical properties of the polar organic solvents on the self-assembly of GAV-9 peptide.     

Functionalized hydroxyethylamine based peptide nanostructures as potential inhibitors of falcipain-3, an essential proteases of Plasmodium falciparum

Self-assembled peptide based nanostructures gained enough popularity due to their easy biocompatibility and numerous potential applications. An excellent model of self-assembly of hydroxyethylamine based peptide nanostructures was synthesized and characterized by DLS and TEM. Spherical nano structures of I and III were observed with particle size ～50 and ～80 nm, respectively. Further, I and III were screened against anti-malarial target, falcipain-3 (FP3), a crucial cysteine protease involved as a major hemoglobinase of Plasmodium falciparum. Interestingly, compound III completely inhibited the activity of FP3. The effective concentration (1.5 μM) of III found to be more potent than I. This biochemical result was substantiated by molecular-docking studies indicating III to be best inhibitor of FP3. This is the first report showing that bis hydroxethylamine based peptide nanostructures could be very effective inhibitor of malarial cysteine proteases.     

Probing the Putative Active Site of YjdL: An Unusual Proton-Coupled Oligopeptide Transporter from E. coli

YjdL from E. coli is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences of YjdL and YdgR, a prototypical E. coli POT, were compared in light of the crystal structure of a POT from Shewanella oneidensis. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes in specificity in terms of uptake inhibition. Most strikingly, changing the YjdL specific Asp392 to the conserved Ser in YjdL obliterated the preference for a positively charged C-terminal residue. Based on this unique finding and previously published results indicating that the dipeptide N-terminus may interact with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance.     

Synthesis and β-sheet propensity of constrained N-amino peptides

The stabilization of β-sheet secondary structure through peptide backbone modification represents an attractive approach to protein mimicry. Here, we present strategies toward stable β-hairpin folds based on peptide strand N-amination. Novel pyrazolidinone and tetrahydropyridazinone dipeptide constraints were introduced via on-resin Mitsunobu cyclization between α-hydrazino acid residues and a serine or homoserine side chain. Acyclic and cyclic N-amino peptide building blocks were then evaluated for their effect on β-hairpin stability in water using a GB1-derived model system. Our results demonstrate the strong β-sheet stabilizing effect of the peptide N-amino substituent, and provide useful insights into the impact of covalent dipeptide constraint on β-sheet folding.     

Self Assembly of Short Aromatic Peptides into Amyloid Fibrils and Related Nanostructures

The formation of amyloid fibrils is the hallmark of more than twenty human disorders of unrelated etiology. In all these cases, ordered fibrillar protein assemblies with a diameter of 7–10 nm are being observed. In spite of the great clinical important of amyloidassociated diseases, the molecular recognition and self-assembly processes that lead to the formation of the fibrils are not fully understood. One direction to decipher the mechanism of amyloid formation is the use of short peptides fragments as model systems. Short peptide fragments, as short as pentapeptides, were shown to form typical amyloid assemblies in vitro that have ultrastructural, biophysical, and cytotoxic properties, as those of assemblies that are being formed by full length polypeptides. When we analyzed such short fragments, we identified the central role of aromatic moieties in the ability to aggregate into ordered nano-fibrillar structures. This notion allowed us to discover additional very short amyloidogenic peptides as well as other aromatic peptide motifs, which can form various assemblies at the nano-scale (including nanotubes, nanospheres, and macroscopic hydrogels with nano-scale order). Other practical utilization of this concept, together with novel β breakage methods, is their use for the development of novel classes of amyloid formation inhibitors.Key Words: Alzheimer''s disease, amyloid disease, molecular recognition, nanostructures, protein aggregation, protein misfolding, self-assembly, type II diabetes     

Charge effects on the fibril-forming peptide KTVIIE: a two-dimensional replica exchange simulation study

The assembly of peptides into ordered nanostructures is increasingly recognized as both a bioengineering tool for generating new materials and a critical aspect of aggregation processes that underlie neurological diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. There is a major problem in understanding how extremely subtle sequence changes can lead to profound and often unexpected differences in self-assembly behavior. To better delineate the complex interplay of different microscopic driving forces in such cases, we develop a methodology to quantify and compare the propensity of different peptide sequences to form small oligomers during early self-assembly stages. This umbrella-sampling replica exchange molecular dynamics method performs a replica exchange molecular dynamics simulation along peptide association reaction coordinates using umbrella restraints. With this method, we study a set of sequence-similar peptides that differ in net charge: K(+)TVIIE(-), K(+)TVIIE, and (+)K(+)TVIIE. Interestingly, experiments show that only the monovalent peptide, K(+)TVIIE, forms fibrils, whereas the others do not. We examine dimer, trimer, and tetramer formation processes of these peptides, and compute high-accuracy potential of mean force association curves. The potential of mean forces recapitulate a higher stability and equilibrium constant of the fibril-forming peptide, similar to experiment, but reveal that entropic contributions to association free energies can play a surprisingly significant role. The simulations also show behavior reminiscent of experimental aggregate polymorphism, revealed in multiple stable conformational states and association pathways. Our results suggest that sequence changes can have significant effects on self-assembly through not only direct peptide-peptide interactions but conformational entropies and degeneracies as well.     

一种溶酶体靶向的原位自组装短肽设计及抗肿瘤活性研究

肿瘤已成为威胁人类生命的一大杀手,目前主要采用手术和放、化疗等手段进行治疗,但由于放、化疗的细胞选择性差、毒副作用明显且易引起肿瘤细胞产生耐受(/药)性,不利于肿瘤的持续治疗,因此亟待研发具有定向定位优势、毒副作用低的新型靶向药物.原位自组装多肽能识别肿瘤部位的特异性高表达物质,在肿瘤部位靶向性聚集形成稳定的纳米结构,...     

One-dimensional self-assembly of a rational designed beta-structure peptide

Fabricating various nanostructures based on the self-assembly of diverse biological molecules is now of great interest to the field of bionanotechnology. In this study, we report a de novo designed peptide (T1) with a preferential beta-hairpin forming property that can spontaneously assemble into nanofibrils in ultrapure water. The nanofibrils assembled by T1 could grow up to tens of microns in length with a left-handed helical twist and an average height of 4.9 +/- 0.9 nm. Moreover, protofilaments and nucleus structures both with a similar height of 1.4 +/- 0.2 nm were observed during fibrilization as well as via sonication of the mature nanofibrils. A typical conformational transition from random coil to beta-structure was observed in association with the fibrilization. Molecular modeling of T1 assemblies displayed that the beta-hairpin molecules organize in a parallel fashion in which the beta-strands align in an antiparallel fashion and each adjoining beta-strand runs left-handed twist at about 2.9 degrees with respect to the one located before it along the fibrillar axis. It also revealed that the maximum thickness of the assembly intermediate, the helical tape structure, is about 1.4 nm and four tapes can further assemble into a fibril with a diameter of about 4.1 nm. Taken together the results obtained by AFM, CD, and molecular modeling, T1 fibrilization probably undergoes a hierarchy approach, in which the aromatic stacking and the electrostatic interactions between the assembled structures are most likely the two major factors directing the one-dimensional self-assembly. Based on these studies, we propose T1 can be used as a model peptide to investigate the beta-sheet based self-assembly process and could be a potential bioorganic template to develop functional materials.     

Stimuli responsive self-assembled hydrogel of a low molecular weight free dipeptide with potential for tunable drug delivery

Bottom-up fabrication by molecular self-assembly is now widely recognized as a potent method for generating interesting and functional nano- and mesoscale structures. Hydrogels from biocompatible molecules are an interesting class of mesoscale assemblies with potential biomedical applications. The self-assembly of a proteolysis resistant aromatic dipeptide containing a conformational constraining residue (DeltaPhe) into a stable hydrogel has been studied in this work. The reported dipeptide has free -N and -C termini. The hydrogel was self-supportive, was fractaline in nature, and possessed high mechanical strength. It was responsive to environmental conditions like pH, temperature, and ionic strength. The gel matrix could encapsulate and release bioactive molecules in a sustained manner. The described hydrogel showed no observable cytotoxicity to the HeLa and L929 cell lines in culture.     

Conformational behaviour of the architectural units of peptides and proteins. Assessment of current understanding by ab initio quantum mechanical methods and refinement of the dipeptide energy surface.

The conformational energy surfaces of analogues of the dipeptide unit of polypeptides and proteins are calculated by ab initio methods using extended basis sets.The calculations are not particularly sensitive to the choice of (extended) basis set.The calculations are shown to support a particular empirical method parameterized with respect to crystal data. Non-hydrogen bonded conformations agree to within 3 kcal mol^?1, even for conformations in which quite considerable degrees of atomic overlap occur.Hydrogen bonded conformations, are, however, in less satisfactory agreement and it is the ab initio calculations which appear to be at fault.A simple correction is applied to the ab initio energy for hydrogen bonded conformations, and with the use of the empirical energy surface a full quantum mechanical conformational energy map is interpolated for the alanyl dipeptide.The effect of flexibility in the peptide backbone is taken into account, and supports recent empirical findings that distortions in valence angles must be considered in calculations of the conformational behaviour of peptides.     

Mechanism of inhibition of proton: Dipeptide co-transport during chronic enteritis in the mammalian small intestine

Amino acids, a critical energy source for the intestinal epithelial cells, are more efficiently assimilated in the normal intestine via peptide co-transporters such as proton:dipeptide co-transport (such as PepT1). Active uptake of a non-hydrolyzable dipeptide (glycosarcosine) was used as a substrate and PepT1 was found to be present in normal villus, but not crypt cells. The mRNA for this transporter was also found in villus, but not crypt cells from the normal rabbit intestine. PepT1 was significantly reduced in villus cells also diminished in villus cell brush border membrane vesicles both from the chronically inflamed intestine. Kinetic studies demonstrated that the mechanism of inhibition of PepT1 during chronic enteritis was secondary to a decrease in the affinity of the co-transporter for the dipeptide without an alteration in the maximal rate of uptake (V_max). Northern blot studies also demonstrated unaltered steady state mRNA levels of this transporter in the chronically inflamed intestine. Proton dipeptide transport is found in normal intestinal villus cells and is inhibited during chronic intestinal inflammation. The mechanism of inhibition is secondary to altered affinity of the co-transporter for the dipeptide.     

Computational design of allosteric ribozymes as molecular biosensors

Nucleic acids have proven to be a very suitable medium for engineering various nanostructures and devices. While synthetic DNAs are commonly used for self-assembly of nanostructures and devices in vitro, functional RNAs, such as ribozymes, are employed both in vitro and in vivo. Allosteric ribozymes have applications in molecular computing, biosensoring, high-throughput screening arrays, exogenous control of gene expression, and others. They switch on and off their catalytic function as a result of a conformational change induced by ligand binding. Designer ribozymes are engineered to respond to different effectors by in vitro selection, rational and computational design methods. Here, I present diverse computational methods for designing allosteric ribozymes with various logic functions that sense oligonucleotides or small molecules. These methods yield the desired ribozyme sequences within minutes in contrast to the in vitro selection methods, which require weeks. Methods for synthesis and biochemical testing of ribozymes are also discussed.