Proteins

Proteins and Peptides

Myoglobin and hemoglobin are important examples of the class of compounds known as proteins, which are linear polymers of between 40 and 10,000 (or more) amino acids. The average molecular weight of an amino acid is about 110 amu. As a result, a modestly sized protein with only 300 amino acids has a molecular weight of 33,000 g/mol, and very large proteins can have molecular weights as high as 1,000,000 g/mol.

Proteins are formed by joining the -CO₂H end of one amino acid with the -NH₂ end of another to form an amide. The -CONH- bond between amino acids is known as a peptide bond because relatively short polymers of amino acids are known as peptides.

The same -CONH- bond forms the backbone of both proteins and synthetic fibers such as Nylon. This raises an interesting question: How do we explain the enormous range of structures and functions of proteins when Nylon has such regular properties?

Nylon has a regular structure that repeats monotonously from one end of the polymer to the other because the monomers from which it is made are symmetrical. The two ends of an amino acid, on the other hand, are different. Each monomer has both an -NH₂ head and a -CO₂H tail. Thus, four different dipeptides can be formed from only two amino acids. Aspartic acid (Asp) and phenylalanine (Phe), for example, can give two symmetrical dipeptidesPhe-Phe and Asp-Asp and two unsymmetrical dipeptides Phe-Asp and Asp-Phe as shown in the figure below. When the full range of amino acids is considered, it is possible to make 400 (20²) different dipeptides, 64 million (20⁶) different hexapeptides, and 10⁵² (20⁴⁰) different proteins that contain only 40 amino acids.

Differences between the structures of even closely related dipeptides such as Asp-Phe and Phe-Asp give rise to significant differences in their properties. The methyl ester of Asp-Phe, for example, has a very sweet taste and is sold as an artificial sweetener under the trade name aspartame.

The ester of the dipeptide with the opposite arrangement of amino acids, Phe-Asp, does not taste sweet and has no commercial value. As the length of the polymer chain increases and the number of possible combinations of R groups increases, polymer chains with an almost infinite variety of structures and properties are produced.

In recent years, a group of naturally occurring peptides that mimic pain-killing drugs such as morphine has been discovered in human brain cells. These enkephalins (from the Greek meaning "in the head") hold the promise of a synthetic pain-killer that is both safe and nonaddictive. One of these enkephalins is a pentapeptide that contains four different amino acids tyrosine (Tyr), glycine (Gly), phenylalanine (Phe), and methionine (Met). The first step in describing the structure of this peptide is to list the amino acids in the order in which they are found on the peptide chain: Tyr-Gly-Gly-Phe-Met. We then have to identify the amino acid at the -CO₂H end of the chain and the amino acid at the -NH₂ end. By convention, proteins are listed from the N-terminal amino acid residue toward the C- terminal end. The structure of this enkephalin is shown in the figure below.

The pentapeptide in the figure below illustrates the perils that face anyone who tries to synthesize peptides or proteins from amino acids.

In order to make this enkephalin in large quantities, we would have to overcome the following problems.

• Peptide bonds are not easy to form. In theory, a carboxylic acid could react with an amine to form an amide.

In practice, carboxylic acids are more likely to react with amines in a simple acid-base reaction to form a salt.

We therefore have to find a way to force the reaction to form the amide.

• Forming a peptide bond is also an uphill process (G^o = +17 kJ/mol_rxn), so a way must be found to drive this reaction forward.
• Because the sequence of amino acids is important, they must be added to the chain one at a time, in a carefully controlled fashion. Thus, a significant entropy factor must be overcome during the synthesis of peptides or proteins.
• The R groups of certain amino acids must be protected during polymerization so that no reactions take place on these side-chains.

In 1984 R. B. Merrifield received the Nobel Prize in chemistry for developing an automated approach to the synthesis of peptides. The first step involves attaching the amino acid that will become the C-terminal residue to an inert, insoluble polystyrene resin. Amino acids are then incorporated, one at a time, by coupling them onto the growing peptide chain. Because the product of each step in this reaction is a solid, it can be easily collected, washed, and purified before the next step in the reaction.

The Merrifield synthesis uses a dehydrating agent known as dicyclohexylcarbodi-imide (DCC) to drive the reaction that forms the peptide bond. To prevent reactions at the wrong site, appropriate blocking groups are added to reactive sites on the side chains of the amino acids before they are polymerized. If we use the symbol B to indicate an appropriate blocking group, the synthesis of a dipeptide can be represented by the following equation.

The blocking group on the N-terminal end of the dipeptide is then removed, and a third blocked amino acid residue is added to give a tripeptide. This process of adding one amino acid at a time is continued until the polypeptide or protein synthesis is complete. The polypeptide or protein chain is then removed by reacting the resin with HBr in a suitable solvent.

When it was first introduced, this process was automated on an apparatus that required about four hours to add an amino acid residue to the peptide chain. Thus, insulin could be synthesized in approximately 8 days, while ribonuclease, with 124 amino acids, required more than a month. The beauty of the Merrifield synthesis is the yield of each step, which is essentially 99%. The synthesis of ribonuclease, for example, took 369 chemical reactions and 11,931 automated steps, and yet still had an overall yield of 17%.

The Primary Structure of Proteins

The primary structure of a protein is nothing more than the sequence of amino acids, read off one at a time, as if printed on ticker-tape. Insulin obtained from cows, for example, consists of two chains (A and B) with the primary structures shown in the figure below. There is more to the structure of a protein, however, than the sequence of amino acids. The polypeptide chain folds back on itself to form a secondary structure. Interactions between amino acid side chains then produces a tertiary structure. For some proteins, such as hemoglobin, interactions between individual polypeptide chains give rise to a quaternary structure.

The Secondary Structure of Proteins

The peptide bond is a resonance hybrid of the two Lewis structures shown below. The Lewis structure on the left implies that the geometry around the carbon atom is trigonal planar and that the carbon atom and its three nearest neighbors lie in the same plane. The Lewis structure on the right suggests a trigonal planar geometry for the nitrogen atom as well. Because the peptide bond is a hybrid of these resonance forms, these six atoms all lie in the same plane.

Since the N-H and C=O bonds are relatively polar, hydrogen bonds form between adjacent peptide chains.

The fact that the six atoms in the peptide bond must lie in the same plane limits the number of ways in which a polypeptide can be arranged in space. By building models, Linus Pauling and Robert Corey discovered two ways in which a polypeptide chain could maximize the hydrogen bonds between peptides. In one of these structures, the chain forms the a-helix shown in the figure below.

The hydrogen bonds between adjacent peptide bonds allow polypeptides to form a right-handed helix. One turn along this helix contains 3.6 amino acid residues.

The other structure is the b-pleated sheet in the figure below.

The polypeptide chain can fold back on itself to form a structure that looks something liked a pleated sheet. Two different b-pleated sheets structures are found in nature that differ in whether the adjacent polypeptide chains run in the same or opposite directions. This drawing shows the antiparallel structure found in silk.

The Tertiary Structure of Proteins

Most proteins have structures that lie between the extremes of ideal -helixes and -pleated sheets because other factors influence the way proteins fold to form three-dimensional structures. Particular attention must be paid to interactions between the side chains of the amino acids that form the backbone of the protein. The figure below shows four ways in which these amino acid side chains can interact to form the tertiary structure of the protein.

Four factors are responsible for the tertiary structure of proteins:

Disulfide (S-S) linkages. If the folding of a protein brings two cysteine residues together, the two -SH side chains can be oxidized to form a covalent S-S bond. These disulfide bonds cross-link the polypeptide chain.

Hydrogen bonding. In addition to the hydrogen bonds between peptide bonds that gives rise to the secondary structure of the protein, hydrogen bonds can form between amino-acid side chains.

Ionic bonding. The structure of a protein can be stabilized by the force of attraction between amino acid side chains of opposite charge, such as the -NH₃⁺ side chain of Leu and the -CO₂^- side chain of Asp.

Hydrophobic interactions. Proteins often fold so that the hydrophobic side chains of the amino acids Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp are buried within the protein, where they can interact to form hydrophobic pockets. These hydrophobic interactions stabilize the structure of the protein.

Human hair is composed primarily of proteins known as the -keratins that are about 14% cysteine. Hair curls as it grows because of the disulfide (S-S) links between cysteine residues on adjacent protein molecules. The first step in changing the way hair curls involves shaping the hair to our satisfaction and then locking it into place with curlers. The hair is then treated with a mild reducing agent that reduces the S-S bonds to pairs of -SH groups. This relaxes the proteins in the hair, allowing them to pick up the structure dictated by the curlers. The -SH side chains on cysteine residues that are now adjacent to each other are then oxidized by the O₂ in air. New S-S linkages form, locking the hair permanently in place; at least until new hair grows.

The a-keratins are divided into two categories, "hard" and "soft," on the basis of the amount of cysteine they contain. The -keratins in skin are soft because they contain relatively small amounts of sulfur, and disulfide cross-links are uncommon. Although hair is classified as a hard keratin, horn and hoof, which contain even more sulfur, are much less pliable because of the extensive disulfide cross-links that form

The Quaternary Structure of Proteins

As we have seen, hemoglobin is the protein that carries O₂ through the bloodstream to the muscles. This protein consists of four polypeptide chains two a-chains that contain 141 amino acids and two b-chains that contain 146 amino acids. Hemoglobin is therefore an example of a protein that has a quaternary structure. It consists of four polymer chains that must be assembled to form the complete protein.

The polymer chains in a quaternary protein are not linked by covalent bonds such as the S-S bonds that hold together the polypeptide chains in insulin. The primary force of attraction between the a- and b-chains in hemoglobin is the result of interactions between hydrophobic substituents on these polymer chains. In other quaternary proteins, hydrogen bonding or ionic interactions between amino-acid side chains on the surfaces of adjacent polymer chains also contribute to the process by which the polymer chains are held together.

The Denaturation of Proteins

Proteins are fragile molecules that are remarkably sensitive to changes in structure. The replacement of a polar Glu residue by a nonpolar Val at the sixth position on the b-chains of hemoglobin, for example, gives rise to the disease known as sickle-cell anemia. The introduction of a hydrophobic Val residue at this position changes the quaternary structure of hemoglobin. The "sticky," nonpolar side chain on a valine residue at this position causes hemoglobin molecules to cluster together in an abnormal fashion, interfering with their function as oxygen-carrying proteins.

Sickle-cell anemia is the result of a change in the way the protein is assembled from amino acids. The structure of a protein can also be changed after it has been made. Anything that causes a protein to leave its normal, or natural, structure is said to denature the protein. Factors that can lead to denaturation include the following.

• Heating, which disrupts the secondary and tertiary structure of the protein. (The changes we observe when we fry an egg result from denaturation caused by heating.)
• Changes in pH that interfere with ionic bonding between amino-acid side chains.
• Detergents, which make nonpolar amino-acid side chains soluble and thereby destroy the hydrophobic interactions that give rise to the tertiary and quaternary structure of the protein.
• Oxidizing or reducing agents that either create or destroy S-S bonds.
• Reagents such as urea (H₂NCONH₂), which disrupt the hydrogen bonds that form the secondary structure of the protein.