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A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural domains. One domain may appear in a variety of evolutionarily related proteins. Domains vary in length from between about 25 amino acids up to 500 amino acids in length. The shortest domains such as zinc fingers are stabilized by metal ions or disulfide bridges. Domains often form functional units, such as the calcium-binding EF hand domain of calmodulin. Because they are self-stable, domains can be "swapped" by genetic engineering between one protein and another to make chimera proteins.

Important tools in determining domains are structural alignment and sequence alignment.

The concept of the domain was first proposed in 1973 by Wetlaufer after X-ray crystallographic studies of hen lysozyme (Phillips, 1966), papain (Drenth et al., 1968) and by limited proteolysis studies of immunoglobulins (Porter, 1973; Edelman, 1973). Wetlaufer defined domains as stable units of protein structure that could fold autonomously. In the past domains have been described as units of:

• compact structure (Richardson, 1981);
• function and evolution (Bork, 1991);
• folding (Wetlaufer, 1973).

Each definition is valid and will often overlap, i.e. a compact structural domain that is found amongst diverse proteins is likely to fold independently within its structural environment. Nature often brings several domains together to form multidomain and multifunctional proteins with a vast number of possibilities (Chothia, 1992). In a multidomain protein, each domain may fulfil its own function independently, or in a concerted manner with its neighbours. Domains can either serve as modules for building up large assemblies such as virus particles or muscle fibres, or can provide specific catalytic or binding sites as found in enzymes or regulatory proteins.

An appropriate example is pyruvate kinase, a glycolytic enzyme that plays an important role in regulating the flux from fructose-1,6-biphosphate to pyruvate. It contains an all-β regulatory domain, an α/β-substrate binding domain and an α/β-nucleotide binding domain, connected by several polypeptide linkers (George and Heringa, 2002a) (see figure, right). Each domain in this protein occurs in diverse sets of protein families.

The central α/β-barrel substrate binding domain is one of the most common enzyme folds. It is seen in many different enzyme families catalysing completely unrelated reactions (Hegyi and Gerstein, 1999). The α/β-barrel is commonly called the TIM barrel named after triose phosphate isomerase, which was the first such structure to be solved (Banner et al., 1975). It is currently classified into 26 homologous families in the CATH domain database (Orengo et al., 1997). The TIM barrel is formed from a sequence of β-α-β motifs closed by the first and last strand hydrogen bonding together, forming an eight stranded barrel. There is debate about the evolutionary origin of this domain. One study has suggested that a single ancestral enzyme could have diverged into several families (Copley and Bork, 2000), while another suggests that a stable TIM-barrel structure has evolved through convergent evolution (Lesk et al., 1989).

The TIM-barrel in pyruvate kinase is 'discontinuous', meaning that more than one segment of the polypeptide is required to form the domain. This is likely to be the result of the insertion of one domain into another during the protein's evolution. It has been shown from known structures that about a quarter of structural domains are discontinuous (Jones et al., 1998; Holm and Sander, 1994). The inserted β-barrel regulatory domain is 'continuous', made up of a single stretch of polypeptide.

Covalent association of two domains represents a functional and structural advantage since there is an increase in stability when compared with the same structures non-covalently associated (Ghelis and Yon, 1979). Other, advantages are the protection of intermediates within inter-domain enzymatic clefts that may otherwise be unstable in aqueous environments, and a fixed stoichiometric ratio of the enzymatic activity necessary for a sequential set of reactions (Ostermeier and Benkovic, 2000).