Peptide crystallization
Main steps:
- Protein purification- Crystallisation
- Peptide crystallisation
Special:
- Programs for crystallography- X-ray detectors
Basic tutorials:
- Chemistry- Protein
- Peptide
- Amino Acids
Xtal community:
- CCP4BBPeptide crystallization background
Peptides are short amino acid chains, usually without preferred conformation in solution. The only cyclic peptides can mountain single conformation. Nevertheless, like many other flexible molecules, peptides can be crystallizing in appropriate conditions. Furthermore, short peptides are usually reasonable stable and do not lose their structure and properties in dried conditions. Therefore, the most common peptide crystallization technique is evaporation.The main goal of peptide crystallization is to produce well-ordered crystals with uniform content. These crystals should be large enough to diffract x-ray with detectable pattern of diffraction, but small enough to avoid any possible distortion. The optimal size for the peptide crystals is about 0.2-1.0 mm.
Peptide purity is very important parameter. Presence of different contaminations can made crystals almost unsuitable for X-ray analysis. Only a few peptides can crystallize from the mixture of different peptides and amino acids. This property sometime used as a peptide purification technique. For most cases the peptide should be about 95% pure.
Some peptides adopt stable conformation with extra compounds, such as metal ions or cations. This is more common for cyclic-peptides like antibiotics (e.g. Valinomycin). In this case it is very important to use proper compounds to obtain crystals with preferred peptide conformations.
Peptide crystallization by evaporation
Evaporation is most common crystallization technique for small organic compounds and peptides, linear and cyclic. The main idea of the evaporation technique is to produce sated solution of the crystallized chemical compound, and slowly increase its concentration. After passing the maximal concentration the chemical compound will precipitate in order to maintain the maximal concentration in the solution. Precipitant can be in amorphous, crystalline or crystal state.
Step-by-step peptide crystallization
- Peptide solution - Solve a small amount of peptide in organic solvent or solvent mixture. Usually, it is necessary to solve 0.01-0.1 mg of peptide in 100-500 µl of organic solvents. It is necessary to make sure, that all peptides are solved.
- Preparation for evaporation - Place the solvent with peptide in the reservoir and seal with some plastic lid or parafilm. Make few needle-thin holes in the lid. Number of holes depends from solvent evaporation rate.
- Evaporation - The optimal evaporation time is 2-5 days. This time can be adjusted by number and size of holes in the lid. During this time it is necessary to maintain constant room temperature and avoid all shaking of the crystallization reservoir.
- Optimization - If after evaporation the precipitated peptide in the reservoir looks like micro-crystals or crystalline, it means that you almost near perfect conditions. Try to change evaporation time. If the crystallization was performed from solvent mixture, try to change the compounds or concentration in this mixture.
- Multiple experiments - The described above crystallization trials should be performed with maximal number of different solvent and possibly with solvent mixtures.