A protein consisting of domains A, B, and C binds a ligand. Which experimental result would provide the best evidence that domain A is involved in binding and domains B and C are not?

Question

A protein consisting of domains A, B, and C binds a ligand.  Which experimental result would provide the best evidence that domain A is involved in binding and domains B and C are not?

Quizplus · Accepted Answer

11ecba2d_0ef1_e05d_a3bf_6759759d8425_MD0008_00 Protein domains are distinct regions in a polypeptide chain that fold independently. In general, each domain carries out a separate function, although one domain may assist another in performing its function. One of the most common functions of a protein domain is to bind a small molecule, called a ligand. The affinity of a protein for a ligand is measured by the dissociation constant, K_d, where a small K_d corresponds to high binding affinity (or a low tendency of the complex to dissociate). In this question, the protein consists of domains A, B, and C. If domain A is the only domain involved in ligand binding, then binding affinity will be unaffected in the absence of domains B and C. Therefore, if a mutant that contains only domain A can bind the ligand with the same K_d as the wild-type protein, this would suggest that only domain A is involved in ligand binding, and that the other domains are not involved. In contrast, if domains B or C are involved in binding, removing them would affect K_d. (Choice B) A decreased K_d corresponds to a higher binding affinity, so if domains B and C alone have a lower K_d for the ligand than the wild-type protein, this would suggest that domain A interferes with ligand binding. (Choice C) An increased K_d corresponds to a lower binding affinity, so if domain A alone has a higher K_d for the ligand than wild-type binding, this would suggest that either domain B, domain C, or both aid in binding. (Choice D) If a mutant that contains only domains B and C cannot bind the ligand, this shows that domain A is required for binding. However, this does not rule out the possibility that domains B or C are also involved. For example, if domains A and B are both required for binding, removing either one would result in no binding. Removing only domain A does not fully address the role (if any) of domains B and C in binding. Educational objective: Protein domains are distinct, independently folding regions of polypeptide chains that, in general, carry out distinct functions. Ligand binding by a protein domain is measured by the dissociation constant K_d. A high K_d corresponds to low affinity for the ligand, and vice versa.

A Protein Consisting of Domains A, B, and C Binds