Proteins are a basic building block of life on earth. They are the molecules that provide structure, produce energy, and allow communication, movement and reproduction within a cell. They, along with carbohydrates, amino acids, and phospholipids, are the key macromolecules that make up organisms. The body of proteins that make up an organism is referred to as its proteome. Proteomics is the study of these proteomes, including the identification of the proteins and their physiological functions.
The study of proteomics had declined in recent years, but the inception of the Human Genome Project ten years ago revived interest for many researchers. Since then, the proteomes of numerous organisms have been determined. The Human Genome Project determined that the human body contains 30,000 genes. This translates to anywhere from 300,000 to one million possible proteins. The importance of proteomics largely lies in drug design and synthesis. Hopefully, new disease markers and drug targets can be identified that will help design products to prevent, diagnose and treat disease. This cannot be accomplished without knowledge of the proteome, however.
A protein is a biological polymer that usually consists of hundreds of amino acid monomers. The general structure of an amino acid is a carbon atom to which four functional groups are bonded. Three of the groups include a hydrogen atom, an amine group, and a carboxylic acid group. The fourth group, or ‘R’ group, is a hydrocarbon chain. This R group is unique to each particular amino acid and is what determines one amino acid from another.
The primary structure of a protein is its linear sequence of amino acids bound by peptide bonds between a nitrogen atom of one monomer and a carbon atom of another. Disulfide bonds between cysteine residues within the molecule stabilize it. This structure determines the secondary, tertiary, and quaternary structures of the protein as well. Proteins are highly specialized, and a single change in an amino acid monomer can result in a completely different function, or no function at all for the protein. Thus, the primary structure is of utmost importance.
The secondary structure of proteins consists of the primary structure formed into two possible regular structures. These are an alpha helix or a pleated sheet. These structures can organize themselves in a repeating fashion or randomly. Disulfide bonds determine the secondary structure, while hydrogen bonding also stabilizes the conformation.
A protein’s tertiary structure is the overall 3-D configuration of the complete protein. Amino acid residues that are far apart in a primary structure can have steric relationships in 3-D form, and the tertiary structure considers this. The tertiary structure is the most thermodynamically stable for the protein in a certain environment; it can change with environmental changes. This is how proteins are denatured.
A protein may consist of several subunits. The quaternary structure of a protein is made up of all of these subunits bound together by electrostatic and hydrogen bonds. Multisubunit proteins are called oligomers and all of the component parts are monomers or subunits. Proteins may also contain non-amino acid functional structures such as a lipid or a carbohydrate.
In order for proteins to be studied, they must first be isolated. In one dimension, the proteins are separated by charge based on their isoelectric points. The migration of different proteins in an electrically charged environment of graduated pH can separate them when their isoelectric points are different. The proteins move toward the pH at which they have no net charge. The main way this has been achieved is through 2-D polyacrylamide gel electrophoresis, or 2-D PAGE. This experiment can achieve the separation of several thousand different proteins in one gel, while high resolution 2-D PAGE can resolve up to 10,000 proteins per gel. Coomassie blue, silver, and SYPRO Ruby Red stains are typically used to visualize the proteins’ migrations.
Mass spectrometry is a technique used for the determination of the mass of a compound; however, it is also useful in protein identification. The spectrometer ionizes the protein, and this charged molecule is sent into an analyzer on the basis of charge repulsion. This analyzer resolves the proteins based on their mass to charge ratio, thus separating them. The detector passes the information to the computer for analysis and identification. Fragmentation can cause problems in protein separation, so ionization methods that minimize formation of fragments are most useful. These methods include matrix-assisted laser desorption/ ionization, or MALDI and electrospray ionization, or ESI.
Once a protein has been isolated, its structure must be determined. Secondary and tertiary protein structures can be found by two methods: X-ray crystallography and nuclear magnetic resonance. Both methods require that the protein be better than 95% pure for the best results, so the isolation methods are extremely important. The experimental technique can include gel or column separation, dialysis, differential centrifugation, salting out, or HPLC. The choice and order of experiments is modified to suit the protein of interest.
In order to study a protein’s structure through X-ray crystallography, it must be crystallized. The most common methods of crystallization are batch methods and vapor diffusion. A supersaturated solution is formed which causes the protein to associate with other protein molecules. The formation of this solution often requires the addition of precipitants such as polyethylene glycol or certain salts. The identification of the amino acids in the protein are again important here, since they can determine the exact reagents and chemical and physical conditions used to crystallize the protein. The crystals are then mouted and snap frozen. This is accomplished by exposing them to cryogenic liquid or gas.
In X-ray crystallography, the crystals are subjected to X-rays containing a heavy metal atom. This method can determine the protein’s secondary and tertiary structure. The X-rays are scattered by the crystal in a pattern unique to the protein. One drawback is that the radiation can damage or backscatter; keeping the crystals supercooled minimizes this side effect and allows the crystals to be stored and reused. A model of the protein is then constructed using the data translated into electron density maps. This method is highly important in drug design, since it is very precise and can reveal crucial structural data.
Nuclear magnetic resonance, or NMR, spectroscopy involves the alignment of nuclear dipoles of a sample in a magnetic field. These dipoles can change orientation back and forth in a magnetic field and absorb and emit energy for each turn. The spectrometer contains an antenna within the magnet; radio waves are pulsed by this antenna through the magnet. The sample absorbs these pulses as energy and then emits them sometime later. This time is measured and stored on the computer. Most often, pulse sequences are used that take advantage of the strong nuclear dipole of the hydrogen nucleus. The information gained from this experiment allows researchers to map the chemical bond connectivity and the spatial orientation of the proteins. This method is particularly useful in determining the function of active sites on enzymes.
NMR spectroscopy has several advantages over X-ray crystallography. One is that NMR requires no crystallization in order for the protein’s structure to be studied. Currently, this method can resolve proteins with molecular weights up to 30,000 Daltons; it is predicted that structures with molecular weights of up to 100,000 Daltons can soon be determined through this method in the coming years. Another advantage is that NMR is sensitive to motions on the millisecond to second range, which can be directly studied. Even motions as small as those of the nanosecond to microsecond scale can be studied indirectly. X-ray crystallography is also an extremely time-consuming process, and the crystalline structures can be difficult to maintain. However, the greatest advantage of NMR over X-ray crystallography is NMR’s ability to reveal the details of specific structural sites without solving the entire structure.
Current studies in proteomics include the research being done by biochemists at the University of Washington in Seattle. These scientists are studying environmental effects on the transcription and translation of the mRNA molecule. This is the nucleic acid that codes for the manufacture of proteins within the cell. In an earlier study, they reported the use of Translational State Array Analysis, or TSAA. This method allowed for the simultaneous study of mRNA level and translation. They chose Saccharomyces cereviciae as their model and arrested it with the temperature-sensitive cdc 15-2 allele. A control was run in which forty-eight mRNA molecules changed upon release from arrest. However, when the temperature of the cdc 15-2 allele was lowered from 37 C to 25 C, fifty-four molecules of mRNA were affected. Therefore, regulating the translational level seems to affect directly the response of yeast cells to external cues.
Another study done recently again involves the use of Saccharomyces cereviciae. This study, completed at Johns-Hopkins University, attempted to investigate whether indexing a proteome according to its C-terminal sequences could be of use in functional classification of proteins. The basis for this experiment is that the protein C-termini are capable of being recognition signatures for many biochemical processes. The extent to which carboxyl terminal sequences are conserved within the proteome is unknown, but this may be related to certain biological functions and therefore has great importance.
The researchers analyzed the terminal sequences of Saccharomyces cereviciae and found that known and unknown terminal sequences existed. This result supports that there may be additional carboxyl terminal signals whose biological functions are not yet known.
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