Protein Crystallography (World of Microbiology and Immunology)
Protein crystallography is a technique that utilizes x rays to deduce the three-dimensional structure of proteins. The proteins examined by this technique must first be crystallized.
When x rays are beamed at a crystal, the electrons associated with the atoms of the crystal are able to alter the path of the x rays. If the x rays encounter a film after passing through the crystal, a pattern can be produced following the development of the film. The pattern will consist of a limited series of dots or lines, because a crystal is composed of many repeats of the same molecule. Through a series of mathematical operations, the pattern of dots and lines on the film can be related to the structure of the molecule that makes up the crystal.
Crystallography is a powerful tool that has been used to obtain the structure of many molecules. Crystallography data was used, for example, in the determination of the structure of the double helix of deoxyribonucleic acid by American molecular biologist James Watson and British molecular biologist Francis Crick in the 1950s. Bacteria and virus are also amenable to x-ray crystallography study. For example, the structure of the toxin produced by Vibrio cholerae has been deduced by this technique. Knowledge of the shape of cholera toxin will help in the tailoring of molecules that will bind to the active site of the toxin. In this way, the toxin's activity can be neutralized. Another example is that of the tail region of the virus that specifically infects bacteria (bacteriophage). The tail is the portion of the bacteriophage that binds to the bacteria. Subsequently, the viral nucleic acid is injected into the bacterium via the tail. Details of the three-dimensional structure of the tail are crucial in designing ways to thwart the binding of the virus and the infection of the bacterium.
Proteins are also well suited to crystallography. The determination of the three-dimensional structure of proteins at a molecular level is necessary for the development of drugs that will be able to bind to the particular protein. Not surprisingly, the design of antibiotics relies heavily on protein crystallography.
The manufacture of a crystal of a protein species is not easy. Proteins tend to form three-dimensional structures that are quire irregular in shape because of the arrangement of the amino acid building blocks within the molecule. Some arrangements of the amino acids will produce flat sheets; other arrangements will result in a helix. Irregularly shaped molecules will not readily stack together with their counterparts. Moreover, once a crystal has formed, the structure is extremely fragile and can dissolve easily. This fragility does have an advantage, however, as it allows other molecules to be incorporated into the crystal during its formation. Thus, for example, an enzyme can become part of a crystal of its protein receptor, allowing the structure of the enzyme-receptor binding site to be studied.
A protein is crystallized by first making a very concentrated solution of the protein and then exposing the solution to chemicals that slowly increase the protein concentration. With the right combination of conditions the protein can spontaneously precipitate. The ideal situation is to have the precipitate begin at one site (the nucleation site). This site acts as the seed for more protein to come out of solution resulting in crystal formation.
Once a crystal has formed it must be delicately transferred to the machine where the x-ray diffraction will be performed. The crystal must be kept in an environment that maintains the crystal throughout the transfer of crystallographic procedures.
The entire process of protein crystallography is delicate and prone to error. Usually many failures occur before a successful experiment occurs. Yet, despite the effort and frustration, the information that can be obtained about protein structure is considerable.
See also Antibody-antigen, biochemical and molecular reactions; Biochemical analysis techniques; DNA (Deoxyribonucleic acid); Laboratory techniques in immunology; Laboratory techniques in microbiology; Molecular biology and molecular genetics; Proteins and enzymes; Vaccine