Self-assembling peptides are a category of peptides which undergo spontaneous assembly into ordered nanostructures. Originally described in 1993, these designer peptides have attracted interest in the field of nanotechnology for their potential for application in areas such as biomedical nanotechnology, tissue cell culturing, molecular electronics, and more.
Effectively self-assembling peptides act as building blocks for various material and device applications. The essence of this technology is to replicate what nature does: to use molecular recognition processes to form ordered assemblies of building blocks capable of conducting biochemical activities.
Background Peptides can serve as sturdy building blocks for a wide range of materials as they can be designed to combine with a range of other building blocks such as lipids, sugars, nucleic acids, metallic nanocrystals, and so on; this gives the peptides an edge over carbon nanotubes, which are another popular nanomaterial, as the carbon structure is unreactive. They also exhibit biocompatibility and molecular recognition; the latter is particularly useful as it enables specific selectivity for building ordered nanostructures. Additionally, peptides have superb resistance to extreme temperature, detergents, and denaturants.
The ability of peptides to perform self-assembly allows them to be used as fabrication tools, which will continue to grow as a fundamental part of nanomaterials production. The self-assembling of peptides is facilitated through the molecules' structural and chemical compatibility with each other. The structures formed demonstrate physical and chemical stability.
An advantage to using self-assembling peptides to build nanostructures in a bottom-up approach is that specific features can be incorporated; the peptides can be modified to serve specific functions. This approach means the final structures are made from the self-integration of small, simple building blocks. This approach is needed for nanoscale structure, as the top-down method of miniaturizing devices using sophisticated lithography and etching techniques has reached a physical limit. Moreover, the top-down approach applies mainly to silicon-based technology and cannot be used for biological developments.
The peptide structure is organized hierarchically into four levels. The primary structure of a peptide is the sequence of the amino acids of the peptide chain. Amino acids are monomer molecules that carry a carboxyl and an amine functional group; a spectrum of other chemical groups are attached to different amino acids, such as thiols and alcohols. This facilitates the wide range of chemical interactions and, therefore, molecular recognitions that peptides are capable of; for designer self-assembling peptides, both natural and non-natural amino acids are used. They link together in a controlled manner to form short peptides, which link to form long polypeptide chains.
Along these chains, the alternating amine (NH) and carbonyl (CO) groups are highly polar, and they readily form hydrogen bonds with each other. These hydrogen bonds bind peptide chains together to give rise to secondary structures. Stable secondary structures include the alpha-helices and beta-sheets. Unstable secondary structures are random loops, turns, and coils that are formed. The secondary structure that is formed depends on the primary structure; different sequences of the amino acids exhibit different preferences.
Secondary structures usually fold, with a variety of loops and turns, into a tertiary structure. What differentiates the secondary structure from the tertiary structure is that the latter includes non-covalent interactions. The quaternary structure combines two or more different chains of polypeptide to form what is known as a protein sub-unit.
The self-assembly process of the peptide chains includes dynamic—reassembly, which occurs repeatedly in a self-healing manner. The types of interactions that facilitate the reassembly of peptide structures include van der Waals forces, ionic bonds, hydrogen bonds, and hydrophobic forces. These forces also facilitate the molecular recognition function that the peptides encompass. These interactions work on the basis of preference dependent on energy properties and specificity.
A range of different nanostructures can be formed. Nanotubes are defined as elongated nano-objects with definite inner holes. Nanofibrils are solid on the inside, as opposed to the hollow nanotubes.
Processing/Synthesis Peptide synthesis can be easily conducted by the established method of solid-phase chemistry in gram or kilogram quantities. The d-isomer conformation can be used for peptide synthesis.
Nanostructures can be made by dissolving dipeptides in 1,1,1,3,3,3-hexafluoro-2-propanol at 100 mg/ml and then diluting it with water for a concentration of less than 2 mg/ml. Multiwall nanotubes with diameters of 80–300 nm, made of dipeptides from the diphenylalanine motif of Alzheimer's β-amyloid peptide are made by this method. If a thiol is introduced into the diphenylalanine then nano-spheres can be formed instead; nanospheres with diameters of 10–100 nm can also be made this way, from a diphenylglycine peptide.
Characterization Atomic force microscopy can measure the mechanical properties of nanotubes. Scanning-electron and atomic-forces microscopy are used to examine Lego peptide nanofiber structures.
Dynamic light scattering studies show structures of surfactant peptides. Surfactant peptides have been studied using a quick-freeze/deep–etch sample preparation method which minimizes effects on the structure. The sample nanostructures are flash frozen at −196 °C and can be studied three-dimensionally, using Transmission electron microscopy.
Using computer technology, a molecular model of peptides and their interactions can be built and studied.
Specific tests can be performed on certain peptides: for example, a fluorescent emission test could be applied to amyloid fibrils by using the dye Thioflavin T, which binds specifically to the peptide and emits blue fluorescence when excited.
Structure
Dipeptides The simplest peptide building blocks are dipeptides. Nanotubes formed from dipeptides are the widest among peptide nanotubes. An example of a dipeptide that has been studied is a peptide from the diphenylalanine motif of the Alzheimer's β-amyloid peptide.
Dipeptides have also been shown to self-assemble into hydrogels, another form of nanostructures, when connected to the protecting group Fluorenylmethyloxycarbonyl chloride. Experiments focusing on the dipeptide Fmoc-Diphenylalanine have been conducted that have explored the mechanism in which Fmoc-diphenylalanine self-assembles into hydrogels via π-π interlocked β-sheets. Phenylalanine has an aromatic ring, a crucial part of the molecule due to its high electron-density, which favors self-assembly where the rings stack and enable the assembly to occur.
Lego peptides / Ionic self-complementary peptides These peptides are approximately 5 nm in size and have 16 amino acids. The class of Lego peptides has the unique characteristics of having two distinct surfaces being either hydrophobic or hydrophilic, similar to the pegs and holes of Lego blocks. The hydrophobic side promotes self-assembly in water and the hydrophilic side has a regular arrangement of charged amino-acid residues, which in turn brings about a defined pattern of ionic bonds. The arrangement of the residues can be classified according to the order of the charges; Modulus I has a charge pattern of , modulus II , and modulus III , and so on. The peptides self-assemble into nanofibers approximately 10 nm long in the presence of alkaline cations or an addition of peptide solution. The fibers form ionic interactions with each other to form checkerboard-like matrices, which develop into a scaffold hydrogel with a high water content of larger than 99.5–99.9% and pores of 10–200 nm in diameter. These hydrogels allow neurite outgrowth and therefore can be used as scaffolds for tissue engineering.
Surfactant peptides Surfactant–like peptides that undergo self-assembly in water to form nanotubes and nanovesicles have been designed using natural lipids as guides. This class of peptides has a hydrophilic head (with one or two charged amino acids such as aspartic or glutamic acids, or lysine or histidine acids) with a hydrophobic tail (with 4 or more hydrophobic amino acids such as alanine, valine, or leucine). The peptide monomers are about 2-3 nm long and consist of seven or eight amino acids; the peptide length can be adjusted by adding or removing acids.Zhao, X., Design of self-assembling surfactant-like peptides and their applications. Current Opinion in Colloid & Interface Science, 2009. 14(5): p. 340-348.
In water, surfactant peptides undergo self-assembling to form well-ordered nanotubes and nanovesicles of 30–50 nm through intermolecular hydrogen bonds and the packing of the hydrophobic tails in between the residues, like micelle formation. Transmission electron microscopy examination on quick-frozen samples of surfactant-peptide structures showed helical open-ended nanotubes. The samples also showed dynamic behaviours and some vesicle "buds" sprouting out of the peptide nanotubes.
Molecular paint or carpet peptides This class of peptides undergoes self-assembling on a surface and form monolayers just few nanometers thick. These types of molecular "paint" or "carpet" peptides are able to form cell patterns, interacting with or trapping other molecules onto the surface. This class of peptides consists of three segments: the head is a ligand part, which has functional groups attached for recognition by other molecules or cell surface receptors; the middle segment is a "linker", allows the head to interact at a distance away from the surface and which also controls the flexibility and the rigidity of the peptide structure; and, at the other end of the linker, a surface anchor where a chemical group on the peptide forms a covalent bond with a particular surface. This class of peptides has the unique property of being able to change molecular structure dramatically. This property is best illustrated using an example. The DAR16-IV peptide, has 16 amino acids and forms a 5 nm β-sheet structure at ambient temperatures; a swift change in structure occurs at high temperature or a change in pH when a 2.5 nm α-helix forms.
Cyclic peptides Extensive research has been performed on nanotubes formed by stacking cyclic peptides with an even number of alternating D and L amino acids. These nanotubes are the narrowest formed by peptides. The stacking occurs through intermolecular hydrogen bonding, and the end product is a cylindrical structure with the amino acid side chains of the peptide defining the properties of the outer surface of the tube and the peptide backbone determining the properties of the inner surface of the tube. Polymers can also be covalently attached to the peptides, in which case a polymer shell around the nanotube can be formed. By applying peptide design, the inner diameter, which is completely uniform, can be specified; the outer surface properties can also be affected by peptide design. Therefore, these cyclic nanotubes can form in a range of different environments.
Property evaluation One should evaluate the properties (mechanical, electronic, optical, magnetic, etc.) of the material that has been chosen and indicate what the major differences would be if the same material were not at nanoscale. Nanotubes formed from dipeptides are stable under extreme conditions. Dry nanotubes do not degrade until 200 °C; nanotubes display exceptional chemical stability at a range of pH and in the presence of organic solvents. This is a marked difference from natural biological systems, which are often unstable and sensitive to temperature and chemical conditions.