Hanna Rapaport

Self-Replicating Amphiphilic β-Sheet Peptides†

Several different non-enzymatic molecular replication systems have been prepared and analyzed, including nucleic acids, fatty acids, peptides, and organic molecules. This research was evidently motivated by the chemists enthusiasm to shine light on plausible scenarios that may have lead to the origin of life on earth and early molecular evolution, and it also provided new opportunities for understanding fundamental principles, such as molecular recognition and autocatalysis. The study of non-enzymatic replication has been expanded recently beyond autocatalysis, to small molecular networks, in which the replication is also a product of template-assisted cross-catalysis. The design of replicating peptides has centered mainly on helical coiled-coil structures, in which monomeric or dimeric peptides, twenty-five to forty amino acids in length, serve as templates for substrate binding and thus for enhanced condensation and replication. However, it has been postulated that shorter peptides with simpler sequences may also serve as templates for self replication, provided that they are able to arrange themselves into unique and welldefined structures. We show herein that rather simple peptides, close analogues of the synthetic amphiphilic Glu(Phe-Glu)n peptides, [27] can form soluble, one-dimensional b-sheet aggregates in water, which serve to significantly accelerate peptide ligation and self replication. It has been postulated and demonstrated that native amyloid fibrils can replicate, albeit with moderate efficiency. Specific design of synthetic peptides can be used to make soluble aggregates with defined structures and higher replication efficiencies. Towards this aim, it has been shown that peptides comprising of repetitive dyads of hydrophilic and hydrophobic amino acid residues tend to adopt b-pleated sheet arrangements. Recently, it was further revealed that the terminal proline residue, which is rigid, does not have a backbone N H group available for hydrogen bonding, and often acts as a b-sheet breaker, can be used to enhance the formation of ordered b-sheet assemblies. Based on this evidence, we have synthesized the sequence Aba-Glu-(PheGlu)5-Pro of peptide 2 in which the N-terminus proline of the b-sheet forming peptide PFE-5 [34] has been replaced by the capping aromatic 4-acetamidobenzoate (ABA; Table 1). The

Cholesterol Monohydrate Nucleation in Ultrathin Films on Water

The growth of a cholesterol crystalline phase, three molecular layers thick at the air-water interface, was monitored by grazing incidence x-ray diffraction and x-ray reflectivity. Upon compression, a cholesterol film transforms from a monolayer of trigonal symmetry and low crystallinity to a trilayer, composed of a highly crystalline bilayer in a rectangular lattice and a disordered top cholesterol layer. This system undergoes a phase transition into a crystalline trilayer incorporating ordered water between the hydroxyl groups of the top and middle sterol layers in an arrangement akin to the triclinic 3-D crystal structure of cholesterol x H(2)O. By comparison, the cholesterol derivative stigmasterol transforms, upon compression, directly into a crystalline trilayer in the rectangular lattice. These results may contribute to an understanding of the onset of cholesterol crystallization in pathological lipid deposits.

Ordered Peptide Assemblies at Interfaces

Molecular systems composed of peptides or proteins can be programmed to yield intriguing and potentially useful supra-molecular architectures. In the past decade peptide self-assemblies at interfaces have been the subject of various studies aiming at formation of molecular structures with predictable patterns and properties. Most of these systems utilized amphiphilic peptides, usually of a particular secondary structure, that self-assemble through non-covalent intermolecular interactions, into two-dimensional, organized supramolecular structures. The interest in design and preparation of self-assembled functional materials is driven by potential benefits to nanotechnology and nanobiotechnology. This review is restricted to amphiphilic peptide assemblies at interfaces studied by grazing incidence X-ray diffraction and atomic force microscopy, geared towards nanometer-scale structural characterizations.

Acidic peptide hydrogel scaffolds enhance calcium phosphate mineral turnover into bone tissue.

Designed peptides may generate molecular scaffolds in the form of hydrogels to support tissue regeneration. We studied the effect of hydrogels comprising β-sheet-forming peptides rich in aspartic amino acids and of tricalcium phosphate (β-TCP)-loaded hydrogels on calcium adsorption and cell culture in vitro, and on bone regeneration in vivo. The hydrogels were found to act as efficient depots for calcium ions, and to induce osteoblast differentiation in vitro. In vivo studies on bone defect healing in rat distal femurs analyzed by microcomputerized tomography showed that the peptide hydrogel itself induced better bone regeneration in comparison to non-treated defects. A stronger regeneration capacity was obtained in bone defects treated with β-TCP-loaded hydrogels, indicating that the peptide hydrogels and the mineral act synergistically to enhance bone regeneration. In vivo regeneration was found to be better with hydrogels loaded with porous β-TCP than with hydrogels loaded with non-porous mineral. It is concluded that biocompatible and biodegradable matrices, rich in anionic moieties that efficiently adsorb calcium ions while supporting cellular osteogenic activity, may efficiently promote β-TCP turnover into bone mineral.

Effect of surface-exposed chemical groups on calcium-phosphate mineralization in water-treatment systems.

Calcium-phosphate-scale formation on reverse osmosis (RO) membranes is a major limiting factor for cost-effective desalination of wastewater. We determined the effects of various organic chemical groups found on membrane surfaces on calcium-phosphate scaling. Langmuir films exposing different functional groups were equilibrated with a solution simulating the ionic profile of secondary effluent (SSE). Surface pressure-area (Langmuir) isotherms combined with ICP elemental analyses of the interfacial precipitate suggested acceleration of calcium-phosphate mineralization by the surface functional groups in the order: PO(4) > COOH ∼ NH(2) > COOH:NH(2) (1:1) > OH > ethylene glycol. Immersion of gold-coated silicon wafers self-assembled with different alkanethiols in SSE solution showed formation of a hydroxyapatite precipitate by X-ray diffraction and ATR-IR analysis. Data showed diverse influences of functional groups on mineralization, implying low calcium-phosphate scaling for uncharged surfaces or surfaces coated with both positively and negatively charged groups. This information is valuable for understanding scaling processes, and for designing of novel low-scaling membranes for water desalination.

Effects of mutations in de novo designed synthetic amphiphilic β-sheet peptides on self-assembly of fibrils

The self-assembly of two similar amphiphilic peptides into fibril structures is described. Molecular dynamic simulations show that both can organize similarly in a monolayer, but in the fibril bilayer, one prefers a single organization while the other forms two conformational variants. This assembly difference correlates well with our experimental results.

Sheet-Like Assemblies of Charged Amphiphilic α/β-Peptides at the Air–Water Interface

There is growing interest in the design of molecules that undergo predictable self-assembly. Bioinspired oligomers with well-defined conformational propensities are attractive from this perspective, since they can be constructed from diverse building blocks, and self-assembly can be directed by the identities and sequence of the subunits. Here we describe the structure of monolayers formed at the air-water interface by amphiphilic α/β-peptides with 1:1 alternation of α- and β-amino acid residues along the backbone. Two of the α/β-peptides, one a dianion and the other a dication, were used to determine differences between self-assemblies of the net negatively and positively charged oligomers. Two additional α/β-peptides, both zwitterionic, were designed to favor assembly in a 1:1 molar ratio mixture with parallel orientation of neighboring strands. Monolayers formed by these α/β-peptides at the air-water interface were characterized by surface pressure-area isotherms, grazing incidence X-ray diffraction (GIXD), atomic force microscopy and ATR-FTIR. GIXD data indicate that the α/β-peptide assemblies exhibited diffraction features similar to those of β-sheet-forming α-peptides. The diffraction data allowed the construction of a detailed model of an antiparallel α/β-peptide sheet with a unique pleated structure. One of the α/β-peptide assemblies displayed high stability, unparalleled among previously studied assemblies of α-peptides. ATR-FTIR data suggest that the 1:1 mixture of zwitterionic α/β-peptides assembled in a parallel arrangement resembling that of a typical parallel β-sheet secondary structure formed by α-peptides. This study establishes guidelines for design of amphiphilic α/β-peptides that assemble in a predictable manner at an air-water interface, with control of interstrand orientation through manipulation of Coulombic interactions along the backbone.

Highly Stable Pleated‐Sheet Secondary Structure in Assemblies of Amphiphilic α/β‐Peptides at the Air–Water Interface

There is growing interest in the design of molecules that undergo predictable self-assembly. Such systems offer the prospect of tuning the properties of materials based on precise tailoring of molecular structure. Progress in this field is driven both by fundamental scientific curiosity and by the numerous potential applications that can be envisioned for “smart” materials. Peptides are attractive as building blocks for self-assembling materials because it is easy to incorporate a wide array of functionality onto the side chain of these molecules because peptides tend to be biocompatible, and the rules that govern peptide self-association are moderately well understood. Bioinspired oligomers with well-defined conformational propensities, called “foldamers”, have comparable advantages for materials applications, and they resist proteolytic degradation. Herein we describe designed amphiphilic oligomers that contain both a-amino acid and b-amino acid residues, which are called “a/b-peptides”. These amphiphilic oligomers are intended to assemble through the formation of hydrogen-bonded sheets at the air–water interface. The two a/b-peptides we examined, bKbE and bEbK, differ in the positions of the ionizable side chains, which are provided by b-homolysine (bhLys) and b-homoglutamic acid (bhGlu) residues, and both contain 11 residues with 1:1 alternation of a and b subunits along the backbone. bKbE Ac-Pro-bhPhe-Val-bhLys-Thr-bhPheVal-bhGlu-Thr-bhPhe-Pro-NH2 bEbK Ac-Pro-bhPhe-Val-bhGlu-Thr-bhPheVal-bhLys-Thr-bhPhe-Pro-NH2 In an extended conformation, this a/b-peptide backbone can engage in interstrand H-bonding analogous to that found in b-sheets formed by a-peptide strands, as illustrated below.

Transparent, conductive, and SERS-active Au nanofiber films assembled on an amphiphilic peptide template

The use of biological materials as templates for functional molecular assemblies is an active research field at the interface between chemistry, biology, and materials science. We demonstrate the formation of gold nanofiber films on β-sheet peptide domains assembled at the air/water interface. The gold deposition scheme employed a recently discovered chemical process involving spontaneous crystallization and reduction of water-soluble Au(SCN)4(1-) upon anchoring to surface-displayed amine moieties. Here we show that an interlinked network of crystalline Au nanofibers is readily formed upon incubation of the Au(iii) thiocyanate complex with the peptide monolayers. Intriguingly, the resultant films were optically transparent, enabled electrical conductivity, and displayed pronounced surface enhanced Raman spectroscopy (SERS) activity, making the approach a promising avenue for construction of nano-structured films exhibiting practical applications.

Characterizing the adsorption of peptides to TiO2 in aqueous solutions by liquid chromatography.

The interactions between titanium oxide (TiO(2)) and flexible peptides, decorated by amine, carboxyl, and phosphoserine functional groups, were characterized using analytical liquid chromatography with various loading and eluting solutions. This approach enabled discernment of the type of intermolecular interactions generated between the peptides and the metal oxide surfaces in addition to unraveling more subtle effects, specific ions, and oxide phase may have on the adsorption. The peptide presenting Lys residues adsorbed to the oxide surface in the presence of Tris buffer and eluted under conditions that indicated its binding via electrostatic interactions at physiological pH values. Upon adsorption to the oxide in the presence of phosphate buffer, the same peptide exhibited stronger electrostatic interactions with the surface, mediated by the buffer phosphate ions. In Tris-buffered saline (TBS), pH 7.4, as the adsorption medium, the peptide with the phosphoserine residues exhibited affinity indicative of coordinative binding to the titanium oxide, whereas a similar peptide decorated by carboxylate groups failed to adsorb. On the basis of differences in the interactions of these peptides with the TiO(2), the efficient separation of the two peptides was demonstrated. A basic amphiphilic peptide, composed mostly of Lys and Leu residues, was found to strongly adsorb to TiO(2) while in helical conformation only, demonstrating the strong impact the secondary structure may have on adsorption to the surface. The methodology presented in this study allows the elucidation of in situ binding mechanism and relative strengths to titanium oxide surfaces at conditions which resemble biologically relevant environments.