Microbiology and Immunology

Protein crystallography WORLD OF MICROBIOLOGY AND IMMUNOLOGY

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with the protein, rather than in solution, drives the trans-

port process.

The outer membrane of Gram-negative bacteria does

contain proteins that participate in the active transport of spe-

cific molecules to the periplasmic space, which separates the

outer and inner membranes. Examples of such transport pro-

teins include the FhuA of Escherichia coliand FepA of this

and other bacteria. This type of active transport is important

for disease processes, as iron can be crucial in the establish-

ment of an infections, and because available iron is normally

in very low concentration in the body.

See alsoBacterial membranes and cell wall; Protein export

PROKARYOTIC REPLICATION•seeCELL CYCLE

(PROKARYOTIC), GENETIC REGULATION OF

PROMED•seeEPIDEMIOLOGY, TRACKING DISEASES WITH

TECHNOLOGY

PROPRIONIC ACID BACTERIA•seeACNE,

MICROBIAL BASIS OF

PROSTHECATE AND NON-PROSTHECATE

APPENDAGES•seeBACTERIAL APPENDAGES

PProtein crystallographyROTEIN CRYSTALLOGRAPHY

Protein crystallography is a technique that utilizes x rays to

deduce the three-dimensional structure of proteins. The pro-

teins 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 mathemat-

ical 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 acidby American molec-

ular 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 choleraehas 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 bacte-

ria. Subsequently, the viral nucleic acid is injected into the

bacterium via the tail. Details of the three-dimensional struc-

ture of the tail are crucial in designing ways to thwart the bind-

ing 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 surpris-

ingly, the design of antibioticsrelies heavily on protein crys-

tallography.

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 mole-

cules 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 bind-

ing site to be studied.

A protein is crystallized by first making a very concen-

trated 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 sponta-

neously precipitate. The ideal situation is to have the precipi-

tate begin at one site (the nucleation site). This site acts as the

seed for more protein to come out of solution resulting in crys-

tal formation.

Once a crystal has formed it must be delicately trans-

ferred to the machine where the x-ray diffraction will be per-

formed. The crystal must be kept in an environment that

maintains the crystal throughout the transfer of crystallo-

graphic procedures.

The entire process of protein crystallography is delicate

and prone to error. Usually many failures occur before a suc-

cessful experiment occurs. Yet, despite the effort and frustra-

tion, the information that can be obtained about protein

structure is considerable.

See alsoAntibody-antigen, biochemical and molecular reac-

tions; Biochemical analysis techniques; DNA (Deoxyribonu-

cleic acid); Laboratory techniques in immunology; Laboratory

techniques in microbiology; Molecular biology and molecular

genetics; Proteins and enzymes; Vaccine

PROTEIN ELECTROPHORESIS•see

ELECTROPHORESIS

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