Structure of Proteins and peptides

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Chapter: Pharmaceutical Drugs and Dosage: Protein and peptide drug delivery

Proteins and peptides consist of simple building blocks called amino acids, which are linked together by peptide bonds.


Structure

Proteins and peptides consist of simple building blocks called amino acids, which are linked together by peptide bonds. A peptide bond is formed by the nucleophilic addition of the primary amine of one amino acid to the electropositive carboxylate carbon of the other amino acid (Figure 25.1). Two amino acids linked together by a peptide bond form a dipeptide; three amino acids linked together by a peptide bond form a tripeptide; and so on. Polypeptides consist of a linear chain of several amino acids. Long chains of amino acids tend to self-associate and fold into three-dimensional conformations depending on their unique amino acid sequence. Specific functions of proteins are often a function of their unique amino acid sequence and the resulting conformation that makes up the protein.


Figure 25.1 Chemical structure of a typical peptide bond. Polypeptides consist of a linear chain of amino acids successively linked via peptide bonds.

As shown in Figure 25.2, a chain of amino acids forming a polypeptide through covalent linkages constitutes a protein or peptide’s primary struc-ture. Spatial folding of a polypeptide chain through noncovalent interac-tions of neighboring amino acids results in the secondary structure, which consists of patterns of structural domains such as α-helices and β-sheets. The surfaces of polypeptide chains, organized into these domains, can further bond with each other through noncovalent interactions of distant amino acids, which give the overall structure to one polypeptide chain, called the tertiary structure. Spatial interaction of more than one polypep-tide chain to form protein is termed the quaternary structure.


Figure 25.2 Illustration of protein structures: (a) primary structure (amino acid sequence), (b) secondary structure (α-helix and β-sheets), (c) tertiary structure (further folding of the secondary structurally folded protein), and (d) quaternary structure (combination of polypeptides).


Amino acids

There are 20 naturally occurring amino acids that form the structural basis of all the proteins and peptides. The chemical structures of these amino acids, along with their abbreviated and one-letter designations, are pre-sented in Figure 25.3. Each amino acid possesses unique physicochemical properties governed by their chemical structure.


Figure 25.3 Chemical structure of the 20 amino acids commonly found in proteins. The amino acids may be subdivided into five groups on the basis of side-chain structure. Their three- and one-letter abbreviations are also listed.

Table 25.2 Hydrophobicity and acidity of amino acids


·           Nineteen amino acids contain an amino (–NH2) and carboxyl (–COOH) group attached to a carbon atom to which various side chains (–R) are connected. This carbon atom is termed α-carbon because it is next to the carboxylate group in the structure. The amino acid proline is unusual; in that, its side chain forms a direct covalent bond with the nitrogen atom of amino group. This is indicated in the higher hydro-phobic character of proline (higher log P, Table 25.2) compared to most other amino acids.

·           The α-carbon has four different groups attached to it and is chiral, except in the case of glycine. This chirality can lead to two optical isomers, l- and d-amino acids, which would be mirror images of each other. Natural amino acids are exclusively l-amino acids.

Amino acids are classified by the acidity (or basicity) and polarity (or hydrophilic/hydrophobic nature). The acidity/basicity is indicated by their ionization constant, the pKa, whereas the polarity is indicated by log P.

These constants are defined by the following equations and are listed for each amino acid in Table 25.2.

An amino acid backbone can involve the ionization of the acid and/or base:

R-COOH ↔ R-COO + H+

R-NH2 + H+ ↔ R-NH3+


pKa = − log Ka

pKb = − log Kb

p Ka + pKb = p K w = 14,

where p Kw is the ionization constant of water

and


Amino acids with low pKa values are acidic, whereas amino acids with high (>7) pK a values (which would correspond to low pKb values) are basic. Amino acids typically have an acidic carboxylate group and a basic amino group, which contribute to its acidity or basicity. In addition, the side chain may also ionize. Thus, there are multiple pKa values associated with an amino acid. However, in a polypeptide chain, the carboxylate and amino groups are covalently bonded to neighboring amino acids (except for the terminal amino acids). In addition, the electron density on the side chain is influenced by the side chains of other spatially close amino acids. Thus, ionization constants of amino acids in a protein are different than those of pure amino acids. Aspartic and glutamic amino acids are considered acidic because of the presence of ionizable carboxylic acid functional groups. Arginine, histidine, and lysine contain basic ionizable side chains and are referred to as basic amino acids.

The hydrophobic character of amino acids as individual molecules is indicated by their log P value (Table 25.2). These values are predominantly influenced by the ionizable carboxylate and amino functional groups. In a protein structure, these functional groups are covalently bonded. Hydrophobicity, in the context of protein surface, is primarily influenced by the protein structure and the interactions of side chains of amino acids with water (Figure 25.4).


Figure 25.4 Relative hydrophobicity of different amino acids estimated based on either their side-chain sequence (scales 1 and 2) or their typical location in a globular protein structure (scales 3 and 4).

Thus, the hydrophobic character of amino acids depends on their micro-environment in the specific protein. In general, the hydrophobic character of an amino acid has been defined by either (a) physicochemical properties of amino acid side chains while ignoring the effects of the carboxylate and the amino groups (scales 1 and 2 in Figure 25.4), or (b) scaling the prob-ability for an amino acid to be found inside or outside a protein structure by examining three-dimensional structures of known proteins (scales 3 and 4 in Figure 25.4). The scaling criteria inherently result in different predic-tions. For example, cysteine typically forms disulfide bonds in proteins, and stable disulfide bonds are generally present on the hydrophobic interior of a globular protein. Thus, cysteine is relatively more hydrophobic by the scaling criterion of its location in a protein.

Primary structure

The primary structure of a protein refers to the sequence of amino acids and the location of disulfide bonds in the constituent polypeptide chain(s) (Figure 25.2). Primary structure determines a protein’s folding and higher levels of structural organization. However, the primary structure generally cannot predict the three-dimensional structure and shape of the proteins in solution.

Secondary structure

Secondary structure can be described as the local spatial conformation of a polypeptide’s backbone, excluding the constituent amino acid’s side chains. Common secondary structural forms are the α-helix and β-sheets (Figure 25.2). The α-helix results from the helical coiling of a stretch of hydrophobic amino acids with the hydrophobic groups facing inside and the hydrophilic groups facing outside the helix. β-sheets, on the other hand, are characterized by side-by-side hydrogen bonding either within the same chain or between two different chains, thus exposing the amino acid func-tional groups to the solvent medium. The chain folding of the secondary structures often arises from cross-linking through hydrogen bonding or disulfide bridges. Generally, α-helices are present in membrane proteins, whereas secreted proteins mostly have β-sheet or irregular structure.

Tertiary structure

Tertiary structure of a protein refers to the exact three-dimensional struc-ture of its constituent polypeptide chain(s) (Figure 25.2). The spatial prox-imity of secondary structural elements determines the tertiary structure of a polypeptide. Spatially close amino acids on the folded (secondary structure) polypeptide chains can form attractive hydrogen bond, ionic, or hydropho-bic interactions, resulting in stabilization of the tertiary structure. Proteins under physiological conditions assume their distinctive tertiary structure of minimum free energy, which is a prerequisite for their biological function.

Quaternary structure

Quaternary structures are the highest level of protein organization that can be achieved by proteins that have more than one noncovalently linked constituent polypeptide chain (Figure 25.2). These polypeptide chains can associate to form dimers, trimers, and oligomers, which constitute the qua-ternary structure of a protein. Almost all proteins that are greater than 100 kDa have a quaternary structure. For example, hemoglobin consists of nonidentical subunits that associate to form a dimer (heterodimer) or a tetramer (heterotetramer); glutathione-S-transferase consists of homotet-ramer (all subunits identical); collagen is a homotrimeric protein; and the enzyme reverse transcriptase is a heterodimer.

The stabilization of higher orders of protein structure by multiple weak bonds is responsible for the flexibility of structure, which is often required for its functionality. For example, enzymes change conformation upon binding of an agonist, and membrane ion channels change conformation to facilitate transport upon ion binding on their surface.

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