Peptide Crystallization

* Please kindly note that our products and services can only be used to support research purposes (Not for clinical use).

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Creative Peptides has accumulated rich experience and expertise in peptide crystallization services. Our understanding of crystal formation and thermodynamics allows us to develop customized, scalable, and peptide crystallization services.

Why do peptide crystallization?

Having a crystalline solid form of a peptide offers clear advantages compared to the amorphous form:

• Significant impurity rejection: Crystallization is sometimes used as a peptide purification technique
• Improved processability: Crystalline forms can be easier to filter, dry, and store, which are crucial factors in manufacturing, transportation, and storage
• Increased stability: Due to higher purity, tighter molecular arrangement, and restricted molecular motion, crystalline materials can provide better physical and chemical stability than liquid solutions or amorphous states.
• Improved characterization: Due to their uniform and ordered structure, crystalline solids are easier to characterize using techniques such as X-ray crystallography
• Enhanced bioavailability: Some crystalline forms of peptides may have better bioavailability that is they are better absorbed and utilised in the body
• Precise dosage: Crystalline peptides allow for more precise dosage measurements, as there can be a known amount of peptide per volume of the solution
• Controlled release: Certain drug formulations may benefit from the controlled and sustained release properties of crystalline solids
• Cost advantage of crystallization when compared to column chromatography methods for purification
• Enhanced drying properties
• Reduced hygroscopicity
• Control of aggregation and gelling

Peptide self-assembly and crystallization

Peptides and their derivatives, consisting of dozens of amino acids, have attracted widespread attention as biological and bio-inspired building blocks owing to their outstanding applications in the construction of advanced functional materials for nanotechnology and biomedicine. In the past decades, many efforts have been devoted to the preparation of such sophisticated structures exhibiting long range order using diverse methodologies, such as X-ray irradiation, phase transformation, solvent thermal annealing, spinning of aligned supramolecular nanotubes and external field-induced alignment. These well-defined nanostructures could further assemble into higher-ordered assemblies, including even nanoscale crystals.

Linear short peptides

The mechanical properties of the hierarchical self-assembly structure of short peptides such as tert-butoxycarbonyl (Boc)-FF were studied. After evaluation of the mechanical properties of the individual crystal assisted by density functional theory calculations, Boc-FF crystals were found to exhibit well-ordered molecular organization stabilized by a network of hydrogen bonding and aromatic interactions alongside unique elastic flexibility. Scanning electron microscopy analyses showed that the crystals were composed of stacked layers bound by weak interactions, which may be responsible for the material being simultaneously strong, tough and flexible. These Boc-FF crystals represent the simplest form of anisotropic self-assembled materials, bearing great potential in applications requiring contradictory mechanical properties. Self-assembling crystalline nanostructures composed of other short peptides, such as PFF, DYF, YFD, 9-fluorenylmethyloxycarbonyl (Fmoc)-GG, acetylated IVE, acetylated LLE-NH2, acetylated LVE, KLVFF and A6K, have also been shown to emerge as a result of hierarchically oriented substructures.

Fig. 1 Hierarchically oriented crystallization of linear short peptides. (Yuan, C., 2019)

Cyclic peptides

Cyclic peptides have attracted much attention in diverse fields ranging from self-assembling nanomaterials to drugs and chemical biology tools owing to their extraordinary high mechanical strength, toughness and elasticity. To this end, researchers have previously reported the hierarchically oriented crystallization of self-assembled fibrous FF networks triggered by aldehyde. The process involves the intramolecular cyclization of linear FF dipeptides and the formation of crosslinked spherical structures, leading to the transition from gel to crystal. The resulting crystals show 3D-ordered organization, thermal stability, and display optical waveguiding properties. The process relies on intramolecular cyclization and kinetically controlled crystallization, which typically takes at least 1 month. However, an alternative solvothermal treatment can expedite the process to 10 minutes. Additionally, the use of formaldehyde increases the thickness of the crystalline nanobelts.

Cyclic peptide nanotubes with different diameters and structures can be obtained through rational design of the chemical structures. Molecular clusters of cyclic peptides with flat and ring-shaped conformations are mainly connected through hydrogen bonds between amide groups. In general, cyclic peptide self-assembly in aqueous media is initiated by an abrupt solubility change of the peptide, followed by an oriented crystallization of the nanotubes.

Amphiphilic peptides

A prominent study on amphiphilic peptide demonstrated the formation of a fibrillar network of Ala6Glu3 (A6E3) stabilized by electrostatic repulsion between charged fibres. Interestingly, additional charges resulting from the reversible ionization of -COOH groups induced by X-ray irradiation enabled the formation of fibre bundles also at the low amphiphilic peptide concentrations (5-10mM), indicating that the increased charge density on the peptide nanofibre surfaces is the main driving force for the formation of the fibre bundles. This has been further evidenced by the similar bundling and orientation in other amphiphilic peptides with distinct sequences (such as VVAAEEGGREDKETV, VVAAEEGGTKREEVD and AAEEGGREDKETV) and the cytoskeleton of cells. Moreover, such filament bundles trapped in a network were responsible for the subsequent crystallization.

Unlike traditional amphiphilic peptides, nucleobase amphiphilic peptides are a novel type of peptide material that integrate multiple advantages of nucleosides, peptides and amphiphilic chemistry. Such molecules possess robust and precise base-pairing interactions, which is beneficial for the hierarchical self-assembly. Researchers have demonstrated that oriented GC dipeptide nucleic acid crystals can be formed and stabilized by stacking interaction and precise base-pairing interactions.

Fig. 2 Hierarchical self-assembly and crystallization of amphiphilic peptides. (Yuan, C., 2019)

Polypeptides

Considering the ubiquitous hierarchical self-assembly in nature, the core polypeptides derived from proteins have been explored as promising building blocks for the study of protein self-assembly, comprising a simpler model system for understanding protein architectures in functional and aberrant biology. A Tau protein-based 26-mer polypeptide fragment has been shown to form laminated amyloid ribbons through lateral assembly of protofilaments.

In addition to charge interactions, the peptide sequence is another crucial factor that influences the final morphology. A synthetic polypeptide has been de novo designed to form β-sheet filaments, which could further self-assemble into flat fibril laminates through lateral association. The height of each fibril is equal to the extended length of the peptide monomer. Moreover, the lamination degree of the fibrils could be controlled by modulating the self-assembly kinetics factors, such as pH and temperature.

How to crystallize peptides?

Crystallizing peptides refers to the process of creating a solid, stable structure of the peptide that can then be studied and analyzed. This is often necessary for research purposes, particularly in the field of biochemistry and molecular biology. Here is a general guide.

Materials:

• Peptide solution
• Precipitant solution (such as PEG, MPD, and ammonium sulfate)
• Crystallization tray and cover slips
• A dark, undisturbed location with a controlled temperature
• Crystal microscope (optional)

Procedure:

• Dissolve your peptide in a small amount of distilled water or buffer as the solvent. The concentration must be high enough for crystallization to occur.
• Prepare your precipitant solution.
• Load your peptide solution into the wells of a crystallization tray.
• Slowly add the precipitant solution to the peptide solution. The precipitant triggers the peptide in solution to come out of solution and form crystals.
• After adding the precipitant solution, cover the tray with a glass or plastic cover slip, secure it, and leave the tray undisturbed in a dark place. You must make sure that the environment where you place it has controlled temperature as changing temperature can disturb the crystallization process.
• Crystals may take anywhere from a few hours to several days to grow so be patient.
• Examine the tray under a crystal microscope over time to monitor the progress of the crystallization. Crystal growth is typically quite slow and could take days.
• Once crystallization has occurred, the crystallized peptide can be harvested for further study or analysis.

Remember that the success rate of crystallization is highly dependent on many factors, such as temperature, purity of the peptide, peptide concentration, the choice of solvent and precipitant, and the rate of evaporation.

What is the main purpose of peptide crystallization?

The prime purpose of peptide crystallization is to study the structure of the peptide at a molecular level. By crystallizing the peptide, scientists are able to use methods like X-ray crystallography to determine the three-dimensional structure of the peptide, including the arrangement of its atoms and its overall shape. This information can be crucial in understanding the peptide's functions, its interactions with other molecules, and potentially its role in diseases, which can aid in drug design and the development of therapeutics.

Peptide crystallization's primary significance remains in the pharmaceutical industry, with its crucial role in drug research and development. By crystallizing peptides, one can deeply understand how peptides and proteins interact with drugs, enabling design and optimize drugs to treat various diseases effectively. Pure peptide crystals could potentially be used in drug delivery systems. They can be engineered to slowly dissolve and release the drug in a controlled manner. Moreover, peptide crystals can also exhibit unique physical properties, such as high thermal stability, mechanical strength, or optical properties, that can be utilized in material science. Over and above, in the realm of industrial production, peptide crystallization aids in manufacturing superior quality products with less impurity, ensuring efficiency and safety in processing.

Overview of our peptide crystallization service

Using a powerful X-ray diffraction platform, we can provide peptide crystallization services to meet the needs of global customers. Our scientists use X-ray crystallography to determine high-resolution structures to help understand their structure and function and support your peptide therapeutics discovery projects.

Fig. 3 A droplet from an optimized crystal growing experiment containing multiple peptide crystals. (Ryan K, 2015)

Peptide crystallization service includes:

Our peptide crystallization service includes the precise isolation, optimization, and crystallization of peptide molecules. Initially, we begin by conducting a comprehensive analysis of the peptide's physiochemical properties. We then determine the optimal conditions for peptide crystallization, which usually involves precise control of the pH, temperature, concentration, and presence of specific additives.

Once the best crystallization conditions are identified, the peptides are allowed to crystallize over a specified period. These crystals are then isolated for further analysis.

Our team uses X-ray crystallography and other advanced techniques to study the structure of the crystallized peptides in detail. This detailed structural information can provide critical insights into the peptide's functionality and its potential applications, especially in drug design and development.

Our service also includes providing comprehensive and detailed reports on the crystallization process, crystal morphology, and analytical data. All of our processes adhere strictly to industry standards and guidelines, ensuring the accuracy and reliability of our results.

Why choose Creative Peptides?

• Professional scientists
• Cost effective
• Fast and reliable
• Tailored solutions
• Comprehensive services
• Excellent customer support

FAQ

1. Why is peptide crystallization important in biomedical research?

Crystallization of peptides allows researchers to study their structure at an atomic level, leading to insights about how they might function in biological systems. These detailed molecular-level images can advance our understanding of diseases and aid in the development of new drugs.

2. What kind of peptides can be crystallized by your service?

Our service is capable of crystallizing a wide range of peptides, from simple small peptides to complex long-chain peptides. Our team of experts will collaborate with you to determine the most appropriate method for your specific needs.

3. How long does the peptide crystallization service take?

The timeframe can vary depending on the specific requirements and complexity of the peptide. However, most peptide crystallization projects can be completed within 2-4 weeks.

4. What is the cost of your peptide crystallization service?

The cost of our peptide crystallization service can vary depending on factors such as the complexity and length of the peptide, as well as any additional analytical services required. We recommend contacting our customer service team for a more accurate quotation.

5. What are the methods of peptide crystallization?

Peptide crystallization requires the synthesis of peptides with TFA removed, and the purity of peptides above 98% as much as possible. Peptides without homologous structure need to be synthesized after the crystallization conditions of ordinary peptides are determined. Because peptide is essentially a mixture (salt+water+peptide+other impurities) and its conformation is unstable, it is not easy to crystallize. Purity and application of peptide:

• >85%: Immunological applications and polyclonal antibody production
• >90%: SAR studies, bioassays
• >95%: In vitro bioassays such as ELISA, enzymology, biological activity
• >98%: NMR, crystallography, sensitive bioassays

References

1. Spencer, R. K.; Nowick, J. S. A newcomer's guide to peptide crystallography. Israel Journal of Chemistry. 2015, 55(6-7): 698-710.
2. Yuan, C., et al. Hierarchically oriented organization in supramolecular peptide crystals. Nature Reviews Chemistry. 2019, 3(10): 567-588.