Dangers of "domain thinking"

Biochemistry/MCB 568 -- Fall 2007
John W. Little--University of Arizona

Bioc/MCB568 Home Page

Back to description of the two-hybrid system

 

Many modern molecular biologists hold the belief that all proteins are composed of a series of "domains", which can be identified by recombinant DNA experiments. It is important to recognize the assumptions behind this belief, and their limitations. The assumptions are these:

1. A domain is a discrete functional and structural unit, such that it folds as a unit and carries out a particular function.

2. Proteins consist of a number of these domains, laid out in a linear array along the polypeptide chain.

3. The properties of a domain are basically the same when this unit is put into a different context (such as in a hybrid protein, for instance in the two-hybrid system).

These assumptions can be faulty in at several ways:

1. Not all proteins have a domain structure.

2. In many proteins, domains exist but they include portions of the polypeptide from different parts of the chain; for example, a domain might be composed of residues 1-100 and 250-350, while residues 101-249 might be a different domain.

3. Sometimes the properties of a domain change when it is taken out of the context of the intact protein. For instance, some proteins contain "autoinhibitory" regions that inhibit the activity of another part of the protein.

Functional tests reveal whether a particular aspect of protein function is retained. As long as it is, one can conclude that the truncated protein retains this activity. The difficulty can be seen with an example. Suppose that deletion 2 is active, while deletion 3 is not. Many workers conclude that this somehow defines a domain for the activity; that the domain ends somewhere between deletions 2 and 3. But this doesn't, in fact, show that there is a domain, only that removing that part of the protein interferes with function.

How else could it work?

1. There could be a domain with several parts, as discussed above.

2. There could be a domain elsewhere in the N-terminal part of the protein, but the deletion simply destabilizes the protein, or causes it to unfold in such a way as to interfere with function.

3. There could be no discrete domain at all.

Bottom line: Many proteins do have well-defined domains, and they can often be identified by this approach. However, one always needs to interpret data from this approach with a good deal of caution!