Higher level structure

Thus far we have concentrated on the primary structure (amino acid sequence) of a polypeptide.

Higher level protein structure can be described at various levels, i.e. secondary, tertiary and quaternary:

• Secondary structure can be described as the local spatial conformation of a polypeptide's backbone, excluding the constituent amino acid's side chains. The major elements of secondary structure are the a-helix and P-strands, as described below.

• Tertiary structure refers to the three-dimensional arrangement of all the atoms that contribute to the polypeptide.

• Quaternary structure refers to the overall spatial arrangement of polypeptide subunits within a protein composed of two or more polypeptides.

2.3.1 Secondary structure

By studying the backbone of most proteins, stretches of amino acids that adopt a regular, recurring shape usually become evident. The most commonly observed secondary structural elements are termed the a-helix and P-strands, which are usually separated by stretches largely devoid of regular, recurring conformation. The a-helix and P-sheets are commonly formed because they maximize formation of stabilizing intramolecular hydrogen bonds and minimize steric repulsion between adjacent side chain groups, while also being compatible with the rigid planar nature of the peptide bonds.

The a-helix contains 3.6 amino acid residues in a full turn (Figure 2.5). This approximates to a length of 0.56 nm along the long axis of the helix. The participating amino acid side chains protrude outward from the helical backbone. Amino acids most conducive with a-helix formation include alanine, leucine, methionine and glutamate. Proline, as well as the occurrence in close proximity of multiple residues with either bulky side groups or side groups of the same charge, tends to disrupt a-helical formation. The helical structure is stabilized by hydrogen bonding, with every backbone C=O group forming a hydrogen bond with the N—H group four residues ahead of it in the helix. Stretches of a-helix found in globular (i.e. tightly folded, approximately spherical) polypeptides can vary in length from a single helical turn to greater than 10 consecutive helical turns. The average length is about three turns.

Figure 2.5 BaLL-and-stick and ribbon representations of an a-helix. Reproduced from Sun, P. and Boyington. 1997. Current Protocols in Protein Science by kind permission of the publisher, John Wiley and Sons

Stretches of a-helix are most often positioned on the protein's surface, with one face of the helix facing the hydrophobic interior and the other facing the surrounding aqueous medium. The amino acid sequence of these helices is such that hydrophobic amino acid residues are positioned on one

Hydrogen bond

Figure 2.6 The ß-sheet. (a) Two segments of ß-strands (antiparallel) forming a ß-sheet via hydrogen bonding. The ß-strand is drawn schematically as a thick arrow. By convention the arrowhead points in the direction of the polypeptide's C terminus. (b) Schematic illustration of a two-strand ß-sheet in parallel and antiparallel modes

antiparallel ß sheet

Figure 2.6 Continued

antiparallel ß sheet

(b) parallel |i sheet

Figure 2.6 Continued face of the helix, whereas hydrophobic amino acids line the other. The transmembrane sections of polypeptides that span biological membranes often display one (or more) a-helical stretches. In such instances, almost all the residues found in the helix display hydrophobic side chains.

P-strands represent the other major recurring structural element of proteins. P-strands usually are 5-10 amino acid residues in length, with the residues adopting an almost fully extended zigzag conformation. Single P-strands are rarely, if ever, found alone. Instead, two or more of these strands align themselves together to form a P-sheet. The P-sheet is a common structural element stabilized by maximum hydrogen bonding (Figure 2.6). The individual P-strands participating in P-sheet formation may all be present in the same polypeptide, or may be present in two polypeptides held in close juxtaposition. P-sheets are described as being parallel, antiparallel or mixed. A parallel sheet is formed when all the participating P-stretches are running in the same direction (e.g. from the amino terminus to the carboxy terminus; Figure 2.6). An antiparallel sheet is formed when successive strands have alternating directions (N-terminus to C-terminus followed by C-terminus to N-ter-minus, etc.). A P-sheet containing both parallel and antiparallel strands is termed a mixed sheet.

In terms of secondary structure, most proteins consist of several segments of a-helix and/ or P-strands separated from each other by various loop regions. These regions can vary in length and shape, and allow the overall polypeptide to fold into a compact tertiary structure. In addition to their obvious role in connecting stretches of regular secondary elements, loop regions themselves often participate/contribute directly to the polypeptide's biological function. The antigen-binding region of antibodies, for example, is largely constructed from six loop regions (Chapter 13). Such loops also often form the active site of enzymes (Chapter 12). One loop structure, termed a P-turn or P-bend, is a characteristic feature of many polypeptides (Figure 2.7).

Hydrogen bond a carbon of amino acid residue 1

a carbon of amino acid residue 1

Hydrogen bond

Figure 2.7 (a) The P-bend or P-turn is often found between two stretches of antiparallel P-strands. (b) It is stabilized in part by hydrogen bonding between the C= O bond and the NH groups of the peptide bonds at the neck of the turn

Figure 2.7 (a) The P-bend or P-turn is often found between two stretches of antiparallel P-strands. (b) It is stabilized in part by hydrogen bonding between the C= O bond and the NH groups of the peptide bonds at the neck of the turn

2.3.2 Tertiary structure

As mentioned previously, a polypeptide's tertiary structure refers to its exact three-dimensional structure, relating the relative positioning in space of all the polypeptide's constituent atoms to each other. The tertiary structure of small polypeptides (approximately 200 amino acid residues or less) usually forms a single discrete structural unit. However, when the three-dimensional structure of many larger polypeptides is examined, the presence of two or more structural subunits within the polypep-tide becomes apparent. These are termed domains. Domains, therefore, are (usually) tightly folded subregions of a single polypeptide, connected to each other by more flexible or extended regions. As well as being structurally distinct, domains often serve as independent units of function. Cell surface receptors, for example, usually contain one or more extracellular domains (some or all of which participates in ligand binding), a transmembrane domain (hydrophobic in nature and serving to stabilize the protein in the membrane) and one or more intracellular domains that play an effector function (e.g. generation of second messengers). Many therapeutic proteins also display several domains. Tissue plasminogen activator (tPA), for example (Chapter 12), consists of five such domains.

2.3.3 Higher structure determination

There are three potential methods by which a protein's three-dimensional structure can be visualized: X-ray diffraction, NMR and electron microscopy. The latter method reveals structural information at low resolution, giving little or no atomic detail. It is used mainly to obtain the gross three-dimensional shape of very large (multi-polypeptide) proteins, or of protein aggregates such as the outer viral caspid. X-ray diffraction and NMR are the techniques most widely used to obtain high-resolution protein structural information, and details of both the principles and practice of these techniques may be sourced from selected references provided at the end of this chapter. The experimentally determined three-dimensional structures of some polypeptides are presented in Figure 2.8.

Figure 2.8 Three-dimensional structure of (a) human interleukin-4, as determined by NMR, and (b) human follicle-stimulating hormone, as determined by X-ray diffraction. Reproduced from protein data bank (www. rcsb.org/pdb, molecule ID numbers 1 ITM and 1 FL7 respectively)
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