Chapter 2 Introduction to Protein Structure

Proteins are the labor force of the cell. Membrane proteins join hands with the fibrous proteins of the cytoplasm and the extracellular matrix to keep cells and tissues in shape, enzyme proteins catalyze metabolic reactions, and DNA-binding proteins regulate gene expression.

Proteins consist of polypeptides: unbranched chains of amino acids with lengths ranging from less than 100 to more than 4000 amino acids. They form complex higher-order structures that are held together by noncovalent interactions, and they can consist of more than one polypeptide. Some proteins can fold into abnormal conformations that cause aggregation. Such abnormal protein aggregates are an important cause of neurodegenerative diseases and other age-related disorders.

This chapter discusses the 20 amino acids that occur in proteins, the noncovalent higher-order structures of proteins, their physical properties, and the diseases related to abnormal protein folding.

Amino acids are zwitterions

All amino acids have an α-carboxyl group, an α-amino group, a hydrogen atom, and a variable side chain R (“residue”) bound to the α-carbon. This structure forms two optical isomers:

Only the L-amino acids occur in proteins.D-Amino acids are rare in nature, although they occur in some bacterial products.

The pK of the α-carboxyl group is always close to 2.0, and the pK of the α-amino group is near 9 or 10. The protonation state varies with the pH (Fig. 2.1). At a pH below the pK of the carboxyl group, the amino acid is predominantly a cation; above the pK of the amino group, the amino acid is an anion; and between the two pK values, the amino acid is a zwitterion (from German zwitter meaning “hermaphrodite”), that is, a molecule carrying both a positive and a negative charge. The isoelectric point (pI) is defined as the pH value at which the number of positive charges equals the number of negative charges. For a simple amino acid such as alanine, the pI is halfway between the pK values of the two ionizable groups. Note that whereas the pK is the property of an individual ionizable group, the pI is a property of the whole molecule.

Figure 2.1 Protonation states of the amino acid alanine. The zwitterion is the predominant form in the pH range from 2.3 to 9.9.

The pK values of the ionizable groups are revealed by treating an acidic solution of an amino acid with a strong base or by treating an alkaline solution with a strong acid. Any ionizable group stabilizes the pH at values close to its pK because it releases protons when the pH in its environment rises, and it absorbs protons when the pH falls. The titration curve shown in Figure 2.2 has two flat segments that indicate the pK values of the two ionizable groups. In the body, the ionizable groups of proteins and other biomolecules stabilize the pH of the body fluids.

Figure 2.2 Titration curve of the amino acid alanine. The two level segments are caused by the buffering capacity of the carboxyl group (at pH 2.3) and the amino group (at pH 9.9).

The titration curves of amino acids that have an additional acidic or basic group in the side chain show three rather than two buffering areas. The pI of the acidic amino acids is halfway between the pK values of the two acidic groups, and the pI of the basic amino acids is halfway between the pK values of the two basic groups (Fig. 2.3).

Figure 2.3 Prevailing ionization states of the amino acids aspartate (A) and lysine (B) at different pH values. The isoelectric points of aspartate and lysine are 2.95 and 10.0, respectively.

Amino acid side chains form many noncovalent interactions

The 20 amino acids can be placed in a few major groups (Fig. 2.4). Their side chains form noncovalent interactions in the proteins, and some form covalent bonds:

1. Small amino acids: Glycine and alanine occupy little space. Glycine, in particular, is found in places where two polypeptide chains have to come close together.

2. Branched-chain amino acids: Valine, leucine, and isoleucine have hydrophobic side chains.

3. Hydroxyl amino acids: Serine and threonine form hydrogen bonds with their hydroxyl group. They also form covalent bonds with carbohydrates and with phosphate groups.

4. Sulfur amino acids: Cysteine and methionine are quite hydrophobic, although cysteine also has weak acidic properties. The sulfhydryl (—SH) group of cysteine can form a covalent disulfide bond with another cysteine side chain in the protein.

5. Aromatic amino acids: Phenylalanine, tyrosine, and tryptophan are hydrophobic, although the side chains of tyrosine and tryptophan can also form hydrogen bonds. The hydroxyl group of tyrosine can form a covalent bond with a phosphate group.

6. Acidic amino acids: Glutamate and aspartate have a carboxyl group in the side chain that is negatively charged at pH 7. The corresponding carboxamide groups in glutamine and asparagine are not acidic but form strong hydrogen bonds. Asparagine is an attachment point for carbohydrate in glycoproteins.

7. Basic amino acids: Lysine, arginine, and histidine carry a positive charge on the side chain, although the pK of the histidine side chain is quite low.

8. Proline amino acid is a freak among amino acids, with its nitrogen tied into a ring structure as a secondary amino group. Being stiff and angled, it is often found at bends in the polypeptide.

Figure 2.4 Structures of the amino acids in proteins.

The pK values of the ionizable groups in amino acids and proteins are summarized in Table 2.1. Most negative charges in proteins are contributed by the side chains of glutamate and aspartate, and most positive charges are contributed by the side chains of lysine and arginine.

Table 2.1 pK Values of Some Amino Acid Side Chains*

Peptide bonds and disulfide bonds form the primary structure of proteins

The amino acids in the polypeptides are held together by peptide bonds. A dipeptide is formed by a reaction between the α-carboxyl and α-amino groups of two amino acids. For example,

Adding more amino acids produces oligopeptides and finally polypeptides (Fig. 2.5). Each peptide has an amino terminus, conventionally written on the left side, and a carboxyl terminus, written on the right side. The peptide bond is not ionizable, but it can form hydrogen bonds. Therefore peptides and proteins tend to be water soluble.

Figure 2.5 Structure of polypeptides. Note the polarity of the chain, with a free amino group at one end of the chain and a free carboxyl group at the opposite end.

Many proteins contain disulfide bonds between the side chains of cysteine residues. They are formed in a reductive reaction in which the two hydrogen atoms of the sulfhydryl groups are transferred to an acceptor molecule:

The disulfide bond can be formed between two cysteines in the same polypeptide (intra-chain) or in different polypeptides (inter-chain). The reaction takes place in the endoplasmic reticulum (ER), where secreted proteins and membrane proteins are processed. Therefore most secreted proteins and membrane proteins have disulfide bonds. Most cytoplasmic proteins, which do not pass through the ER, have no disulfide bonds.

The enzymatic degradation of disulfide-containing proteins yields the amino acid cystine:

The covalent structure of the protein, as described by its amino acid sequence and the positions of disulfide bonds, is called its primary structure.

Proteins can fold themselves into many different shapes

The peptide bond is conventionally written as a single bond, with four substituents attached to the carbon and nitrogen of the bond:

A C—N single bond, like a C—C single bond, should show free rotation. The triangular plane formed by the O=C—Cα1 portion should be able to rotate out of the plane of the Cα2—N—H portion. Actually, however, the peptide bond is a resonance hybrid of two structures:

Its “real” structure is between these two extremes. One consequence is that, like C=C double bonds (see Chapters 12 and 23), the peptide bond does not rotate. Its four substituents are fixed in the same plane. The two α-carbons are in trans configuration, opposite each other.

The other two bonds in the polypeptide backbone, those involving the α-carbon, are “pure” single bonds with the expected rotational freedom. Rotation around the nitrogen—α-carbon bond is measured as the Φ (phi) angle, and rotation around the peptide bond carbon—α-carbon bond as the ψ (psi) angle (Fig. 2.6). This rotational freedom turns the polypeptide into a contortionist that can bend and twist itself into many shapes.

Figure 2.6 Geometry of the peptide bond. The Φ and ψ angles are variable.

Globular proteins have compact shapes. Most are water soluble, but some are embedded in cellular membranes or form supramolecular aggregates, such as the ribosomes. Hemoglobin and myoglobin (see Chapter 3), enzymes (see Chapter 4), membrane proteins (see Chapter 12), and plasma proteins (see Chapter 15) are globular proteins. Fibrous proteins are long and threadlike, and most serve structural functions. The keratins of hair, skin, and fingernails are fibrous proteins (see Chapter 13), as are the collagen and elastin of the extracellular matrix (see Chapter 14).

α-Helix and β-pleated sheet are the most common secondary structures in proteins

A secondary structure is a regular, repetitive structure that emerges when all the Φ angles in the polypeptide are the same and all the ψ angles are the same. Only a few secondary structures are energetically possible.

In the α-helix (Fig. 2.7), the polypeptide backbone forms a right-handed corkscrew. “Right-handed” refers to the direction of the turn: When the thumb of the right hand pushes along the helix axis, the flexed fingers describe the twist of the polypeptide. The threads of screws and bolts are right-handed, too. The α-helix is very compact. Each full turn has 3.6 amino acid residues, and each amino acid is advanced 1.5 angstrom units (Å) along the helix axis (1 Å = 10^–1 nm = 10^–4 μm = 10^–7 mm). Therefore a complete turn advances by 3.6 × 1.5 = 5.4 Å, or 0.54 nm.

Figure 2.7 Structure of the α-helix.

The α-helix is maintained by hydrogen bonds between the peptide bonds. Each peptide bond C—O is hydrogen bonded to the peptide bond N—H four amino acid residues ahead of it. Each C—O and each N—H in the main chain are hydrogen bonded. The N, H, and O form a nearly straight line, which is the energetically favored alignment for hydrogen bonds.

The amino acid side chains face outward, away from the helix axis. The side chains can stabilize or destabilize the helix, but they are not essential for helix formation. Proline is too rigid to fit into the α-helix, and glycine is too flexible. Glycine can assume too many alternative conformations that are energetically more favorable than the α-helix.

The β-pleated sheet (Fig. 2.8) is far more extended than the α helix, with each amino acid advancing by 3.5 Å. In this stretched-out structure, hydrogen bonds are formed between the peptide bond C—O and N—H groups of polypeptides that lie side by side. The interacting chains can be aligned either parallel or antiparallel, and they can belong either to different polypeptides or to different sections of the same polypeptide. Blanketlike structures are formed when more than two polypeptides participate. The α-helix and β-pleated sheet occur in both fibrous and globular proteins.