Protein crystallization

Protein crystals are investigated in protein, they are made from purified protein and large amounts of water. Crystal protein in a single protein at the lattice points of the lattice are arranged exactly the same. The base of the crystal is then made up to a few thousand atoms.


While one could relatively quickly explain the structure in crystals of simple chemical compounds built with the fledgling method of X-ray structural analysis (NaCl 1913, benzene 1928), appeared to great experimental difficulties in proteins because they consist of thousands of atoms. The first difficulty was the isolation, purification and crystallization of proteins. James Batcheller Sumner This was the first time the enzyme urease in 1926 and at the protein concanavalin A and B from the jack bean. The application of protein crystallization as a general method John Howard Northrop was able to show, for example, on the basis of pepsin in 1929. Both researchers were awarded the Nobel Prize in Chemistry for these developments in 1946.

Not until the early 1930s that the British physicist, crystallographer and science historian John Desmond Bernal and Dorothy Crowfoot Hodgkin his co-worker ( in 1964 alone received the Nobel Prize in Chemistry ), to obtain protein crystals sharp diffraction images. To determine from these diffraction patterns, the three-dimensional protein structure, however, a large computational effort was required, which was only easier to cope with the development of the computer. The first structure of a protein ( myoglobin) was elucidated by John Cowdery Kendrew with the help of X-ray crystallography in 1958 (Nobel Prize in Chemistry in 1962, together with Max Perutz, who had developed the method counts). The recovery of proteins was facilitated by the advent of methods for the recombinant production of recombinant proteins in the 1980s. Previously presented the purification and characterization of a single protein or about the extent of biochemical thesis dar. By genetic engineering methods, many proteins were produced and purified in significantly increased quantities, which lessened the burden for cleaning. Meanwhile, tens of thousands of proteins and protein complexes - even large particles such as ribosomes ( for the first time by Ada Yonath ) and viruses - crystallized and characterized structurally ( see Web RCSB PDB).


Protein crystals occur only very rarely in the cytoplasm, viruses crystalline aggregates are likely to find. The water of crystallization of the protein crystals occupies about 30-70% of the crystal volume, and is partly situated in a quasi- liquid form in the voids between the protein molecules before. Protein crystals are therefore much more sensitive as compared to ionic or molecular crystals, what both the loss of water, and the poor mechanical properties relates to (for example, the very low hardness). The large voids between the protein molecules can be illustrated by the fact that, for example, a colorless lysozyme crystal is solid blue colored by addition of methylene blue, the dye but, after conversion into a dye- free medium, slowly releases again by diffusion (analogous zeolites ). This characteristic of protein crystals are also utilized in the elucidation of protein structure by crystal structure analysis by X-ray diffraction by ( among other things, for example, uranium, mercury ) manufactures heavy-atom derivatives by soaking the crystals in appropriate heavy metal salt solutions. Due to the chiral nature of the naturally occurring proteins crystallize only in the chiral 65 of the 230 possible crystallographic space groups, which have no mirror planes or inversion centers.


To obtain sufficient protein, proteins are often first today overexpressed (usually into E. coli or yeast ), as the purification of proteins is much simpler and is also very easily get to large quantities of protein.

The basic prerequisite for the production of protein crystals are sufficient amounts of high quality proteins (proteins can be by means of combination of precipitation methods, chromatography or preparative electrophoresis separated from other proteins). The crystallization conditions can be found, then, by highly concentrated protein solutions ( 2-20 mg protein / mL) with various buffer solutions, which are usually very high concentrations of salts (eg ammonium sulfate), alcohols (for example contain ethanol and methyl pentanediol ) or polyethylene glycol ( PEG), mixed in small drops with volumes in the nanoliter to microliter range and left to stand over days to weeks and months at a constant temperature.

Thus, nuclei are formed, the protein - precipitant mixture must be in Nukleationsbereich, ie in the phase diagram in the supersaturated region. The following methods are concerned, this area is gradually approaching:

  • Hanging or sitting drop method: a drop of the protein solution with a low concentration of the precipitating agents is at the top or side of a vessel via a solution having a high concentration of the precipitating agent. Via the gas phase gradual diffusion of the solvent ( water) takes place, which leads to a saturation in the drops.
  • Diffusion across the phase boundaries between protein solution and the precipitating agent solution may be placed in a capillary of a common phase boundary contact with each other. The precipitating agent with its much smaller particle diffuses through the interface into the protein solution.

In the batch process, the solution must already be in Nukleationsbereich. The sample and precipitant mixture are mixed with an insulating layer to one another to a drop of oil.

For the crystal structure determination are needed crystals ( corresponding crystals are visible on the pictures 1 and 2) - much more common, however, as these arise an amorphous precipitate ( precipitate ) or even crystals, which are not suitable for such a study ( Photos 4 and 5). If even a small protein crystals to grow, which is a great success, because then the crystallization conditions can be optimized. The pH is critical for the solubility parameter, " salting in ", " salting out", ionic strength, organic solvent ( dielectric constant ) and the temperature. The crystallization parameters to be tested at different temperatures. However, there are also several proteins which can not be crystallized, including the majority of membrane proteins have been.

Occasionally observed in its crystallization approaches the formation of quite peculiar crystals, such as a butterfly crystal ( Photo 5 ).


Using the X-ray diffraction, both protein crystals (see Laue method ) and crystalline protein powders are studied (see Debye- Scherrer method ).

Potential Protein crystals are usually frozen prior to examination in the X-ray diffractometer in liquid nitrogen and then mounted in a gas stream at about -170 ° C, to reduce the damage to the crystal protein of the interaction with X-rays. Stray crystals not mean that it is protein crystals, as often crystallize out of the buffer solutions and salts. But these can be distinguished by means of an X-ray investigation of protein crystals, because both produce very typical, different scattering patterns.

For the analysis by X-ray diffraction single crystals of high quality and purity are (depending on the method used ) is required, the preparation is very expensive. Therefore nowadays are increasingly finding high- throughput screening and automated methods in research and industry application, to clean a large number of proteins and to test crystallization conditions. Some stations ( beamlines ) at synchrotron facilities that work with high-intensity X-rays are now equipped with robots that analyze the Diffraktionsverhalten of protein crystals, select appropriate specimens for structure determination and, if appropriate measure full Diffraktionsdatensätze.