Proteins

Properties of Amino Acids

The English language consists of thousands of words, created from any of twenty-six letters arranged in a precise order. In an analogous fashion, proteins are made up of twenty common amino acids in a precise order dictating the protein's structure and function. Every amino acid has a common structure, in which a central carbon is covalently bonded to a carboxyl group (COOH), an amino group (NH₂), a hydrogen, and a variable "R" group.

The chemical properties of the R group are what give an amino acid its character. The R group can be hydrophilic (attracted to water and other polar molecules) or hydrophobic (attracted to nonpolar molecules and repelled by water or other polar molecules). Hydrophilic R groups can have basic charges, as in the amino acid valine, or acidic, as in glutamic acid, or they may even be an uncharged polar group such as-OH (alcohol) or-NH₂ (amino), as in serine. A nonpolar or hydrophobic R group can be a hydro-carbon chain, as in leucine. There are also three special amino acids: cysteine, glycine, and proline. Cysteine has a reactive sulfhydryl R group that forms disulfide bridges (S-S) between regions of the protein chain. These bridges increase toughness and resistance to unfolding of the protein structure. Glycine is the smallest amino acid, with hydrogen as its R group, and it fits into tight places within a protein's structure. Proline has a cyclic ring involving the central carbon, and it causes kinks to occur in a protein chain. Both proline and glycine are common at the corner of turns in the protein foldings.

Primary Structure

The unique sequence of amino acids in a protein is termed the primary structure. When amino acids form a protein chain, a unique bond, termed the peptide bond, exists between two amino acids. The sequence of a protein begins with the amino of the first amino acid and continues to the carboxyl end of the last amino acid.

The unique sequence of amino acids results from the translation of codons present in messenger RNA (mRNA). The mRNA, in turn, is a complementary copy of the gene that codes for that protein. Protein structure and function can change when "misspellings" occur in the order of amino acids during their transcription and translation. Sickle-cell hemoglobin, for example, is "misspelled" in only one amino acid; the sixth amino acid in the beta chain, where a valine is substituted for a glutamic acid. This occurs because the codon for valine, GUG, has replaced the codon for glutamic acid, GAG. This change from acidic to basic amino acid causes the hemoglobin molecules to stick to one another, forming long chains and blocking oxygen binding. These chains of hemoglobin precipitate in the cell, causing the red blood cells to assume a sickle shape. All of these structural and functional changes occur because of the mutation in the hemoglobin gene and a "misspelling" in the hemoglobin's amino acid sequence.

Secondary Structure and Motifs

The secondary structure of proteins is due to foldings that occur within their structure. These foldings are either in a helical shape, called the "alpha-helix" (which was first proposed by Linus Pauling), or a beta-pleated sheet shaped similar to the zig-zag foldings of an accordion. The turns of the alpha-helix are stabilized by hydrogen bonding between every fourth amino acid in the chain. The alpha-helix can cover specific regions of the protein or it may involve the entire protein, as in the alpha-keratin found in claws and horns. The two sides of the alpha-helix may differ in polarity, with hydrophilic R groups projecting to the lining of the channel, while hydrophobic R groups project to the outside of the channel, where they embed in the hydrophobic membrane. This structure is exemplified in membrane channel proteins, proteins that channel ions across from one surface to another. The beta-pleated sheet is formed by folding successive planes. Each plane is five to eight amino acids long. The folds are stabilized by hydrogen bonding. The strength observed in silk fibers is due to their stacks of beta-pleated sheets.

Combinations of secondary structure form "motifs." A coiled-coil motif is common among proteins that associate with the DNA helix. The helix-loop-helix motif is a knobby structure, and the zinc finger projects outward like its name. These last two motifs allow associations between RNA and proteins that form the basis of their interactions.

Tertiary Structure and Protein Domains

Domains are large functional regions of the protein, such as an enzyme's active site, which binds the substrate to the enzyme. Myoglobin, the muscle protein that stores and releases oxygen, contains several alpha-helices wound around a central crevice. It is in this central crevice that the O₂ molecule binds. Just as words take on their meanings when completed, the functional domains unite to form the overall purpose of a protein. For example, a membrane protein stabilizes itself by anchoring itself with a hydrophilic cytoplasmic domain, then weaves its alpha-helices throughout the membrane domain and projects its carbohydrate hydrophilic side chains into the extracellular surface domain. Such membrane proteins often act as receptors, important for receiving signals such as hormones, or work in the immune system to recognize infected cells.

The local foldings, evident in secondary structure, then combine into a single polypeptide chain. This chain is called the tertiary structure, or conformation. For example, the pancreatic enzyme ribonuclease, which aids in digestion of RNA in the diet, consists mainly of beta sheet folds, with three small alpha-helical regions. Tertiary structure is often stabilized by disulfide bonds between adjacent cysteine in different regions of the protein. For example, the tertiary structure of ribonuclease contains four disulfide bonds, located at specific sites. The stability of the tertiary structure of proteins is destroyed by toxic heavy metals such as mercury. Concentrations of mercury in the environment, for example, result in the displacement of hydrogen on the sulfur atom (SH), thereby blocking functional disulfide bonds.

Several other weak, noncovalent interactions also help stabilize tertiary structure. These noncovalent interactions can be disrupted by heating a protein or exposing it to extremes in pH (acidity or alkalinity), which alters the charge of polar groups on the amino acids. Such disruptions cause the protein to unfold, often exposing hydrophobic groups and leading to precipitation (clumping together) of the protein. If these disruptive factors are removed, some proteins can refold to their original conformation. This ability to refold confirms that protein folding is a self-assembly process that is dependent upon the sequence of amino acids.

Quaternary Structure

Some proteins need to functionally associate with others as subunits in a multimeric structure. This is called the quaternary structure of the protein. This can also be stabilized by disulfide bonds and by noncovalent interactions with reacting substrates or cofactors. For example an antibody consists of two "light" polypeptide chains covalently linked to two longer "heavy" chains, forming a Y-shaped molecule with each branch able to bond to an identical antigen. The protein subunits of the single-stranded binding protein of Escherichia coli bind to DNA only as a tetramer (a multimeric form), acting to stabilize the separated DNA strands during replication.

Another excellent example of quaternary structure is that of hemoglobin. Adult hemoglobin consists of two alpha subunits and two beta subunits, held together by noncovalent interactions. Each of the four subunits contains a heme group that binds an oxygen molecule, O₂. This binding of oxygen is a cooperative process whereby the binding of one oxygen molecule occurs slowly, but once achieved then speeds the binding of the remaining three oxygen molecules. The fourth oxygen molecule binds 300 times faster than the first oxygen molecule. This cooperativity assures that maximum oxygen is captured and retained as it enters into the capillaries within the lungs.

The unloading of oxygen is also facilitated by cooperativity, such that after one oxygen molecule is released, the other three soon follow. This assures that the tissues will receive maximum oxygen once it is delivered. Alpha-hemoglobin by itself, or tetramers of all beta subunits, also bind oxygen, but not with the same cooperativity. Such evidence indicates that there is some form of molecular interaction between the subunits of the tetramer of adult hemoglobin.

Signal Sequences in Protein Synthesis

Protein must be delivered to the proper destination in the cell to function properly. Signal sequences within the protein itself act like "zip codes" to ensure correct delivery. The synthesis of secreted proteins like insulin and of proteins that will be integral to the plasma membrane occurs at a ribo-some tethered to the endoplasmic reticulum, which is a system of membranes that transport materials within cells. The peptides formed there are then translocated into the lumen, or channel, of the endoplasmic reticulum, where they will be formed into a polypeptide chain. This translocation occurs because of a specific signal sequence that is formed by the first twenty or so amino acids in the protein. The core of this sequence consists of ten to fifteen amino acids that have hydrophobic side chains such as alanine, leucine, valine, isoleucine, and phenylalanine, which are usually cleaved from the protein later on. The nascent polypeptide chain is guided along this path by a signal receptor protein.

Proteins targeted for internal cellular functions are synthesized on ribosomal assemblages that float free in the cytoplasm. Such proteins also have their signal sequences. Proteins destined for the cell's nucleus have a specific nuclear signal sequence consisting of a small series of basic amino acids such as arginine and lysine bounded by proline. This nuclear signaling sequence can be located anywhere in the protein's sequence as long as it projects outward from the three-dimensional tertiary structure. Signal sequences for proteins targeted to be part of organelles such as the mitochondria and chloroplasts are anywhere from twenty to seventy amino acids long and are mostly hydrophilic. This charged nature allows easy travel through the hydrophilic cytoplasm to the organelle.

Molecular Chaperones

Although the folding of the protein into its tertiary structure is determined by the primary order of amino acids, the process of folding occurs with the assistance of molecular chaperone proteins. These molecular chaperones often have pockets or tunnels that envelop the nascent polypeptide. This enveloping allows the folding of the protein to occur unhindered by unwanted interaction with other cellular components.

Chemical Modification and Processing of Proteins

Most proteins are structurally altered after synthesis through chemical modification or processing. These alterations help the cell determine a protein's fate, such as whether that protein is active or inactive, how long the protein will function, and to some degree the location where that protein will function. Chemical modifications, which are additions of chemical groups to the R groups in the amino acids, are made after translation. Such modifications may include the attachment of a phosphate group (phosphorylation) to the alcohol group on the amino acids of serine, threonine, or tyrosine. The amino acid proline in proteins such as collagen is often hydroxylated, which means that an alcohol group is attached. Other amino acids with amino groups in their R region, such as lysine or arginine, may be chemically modified through methylation, which is the addition of a methyl group (-CH₃), or through acetylation, in which an acetyl group (-CH₃CO) is added. Larger modifications, such as the addition of a carbohydrate group, occur to create glycoproteins in specialized organelles termed Golgi apparati.

Modifications change the charge of the protein, and often cause a change in the protein's activity level. For many DNA-associated proteins their regional acetylations cause them to "loosen" their grip on the DNA helix, thereby enabling transcription factors to enter, signaling gene activation. A cascade of internal protein phosphorylation (successive additions of a phosphate group) is a common mechanism for carrying a hormone's message from the membrane, where it docks into the cell and induces a metabolic change inside the target cell.

Processing results in cutting off specific parts of the protein (cleavage). Many digestive proteins such as pepsin and hormones such as insulin are processed. Pepsin, which is a digestive protein secreted into the lumen of the stomach, remains in an inactive form until stomach acid is also secreted. The timing of the acid secretion, pepsin activation, and entry of food coincide so that pepsin's activity will be directed toward the food and not the wall of the stomach.

Conformational Changes in Protein Structure

As noted above, a protein's activity can be regulated when it undergoes a change in its conformation. A dramatic and extensively studied model of protein conformational change is that of the Na⁺/K⁺ ATPase pump. This is an integral membrane protein with one side facing the exterior of the cell and the other facing the cytosol. It is used for the specific transport of sodium or potassium across the membrane, and one of its most important functions is the repolarization of a nerve fiber after it "fires."

The first step in the transport process is the binding of three Na⁺ (sodium) ions to the inside face of the protein. This is followed by protein phosphorylation using ATP, which causes the protein to change its conformation. This moves the sodium ions from the cytosol to the exterior. This conformational change also opens up exterior binding sites, which tightly bind two potassium ions outside the cell. Following the potassium binding, the protein is dephosphorylated, losing its recently added phosphate group. This dephosphorylation then changes the protein back to the original conformation, causing the protein to loosen its binding of potassium and deliver those two ions to the cytosol. This process demonstrates that protein structure can be reversibly changed. The net result is that the inside of the cell develops a slight negative charge compared to the outside. The disruption of this "polarized" state constitutes nerve cell firings, which allow the cells of the nervous system to communicate with one another.

Proteomics

Proteomics is a new field of study that seeks to describe which proteins are expressed in a cell, when they are expressed, what consequences result from their expression, and how they fit into biochemical pathways. The first step in the study of proteomics is to define the language of protein structure. The field of proteomics promises to bring a complex understanding to the role of proteins in living cells.

SEE ALSO CELL, EUKARYOTIC; CHAPERONES; GENETIC CODE; HEMOGLOBINOPATHIES; IMMUNE SYSTEM GENETICS; MUTATION; NUCLEASES; PROTEOMICS.

Paul K. Small

Bibliography

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Lodish, Harvey, et al. Molecular Cell Biology, 4th ed. New York: W. H. Freeman, 2000.

Sadava, David E. Cell Biology: Organelle Structure and Function. Boston: Jones and Bartlett, 1993.

Stryer, Lubert. Biochemistry, 3rd ed. New York: W. H. Freeman, 1988.