A series of point mutations reveal interactions between the calcium-binding sites of calmodulin

✍ Scribed by Melissa A. Starovasnik; Rachel E. Klevit; Dai-Rong Su; Kathy Beckingham

• Book ID: 105356011
• Publisher: Cold Spring Harbor Laboratory Press
• Year: 2008
• Tongue: English
• Weight: 932 KB
• Volume: 1
• Category: Article
• ISSN: 0961-8368
• DOI: 10.1002/pro.5560010206

✦ Synopsis

Abstract

Calmodulin is a member of the “EF‐hand” family of Ca^2+^‐binding proteins. It consists of two homologous globular domains, each containing two helix‐loop‐helix Ca^2+^‐binding sites. To examine the contribution of individual Ca^2+^‐binding sites to the Ca^2+^‐binding properties of CaM, a series of four site‐directed mutants has been studied. In each, the glutamic acid at position 12 in one of the four Ca^2+^‐binding loops has been changed to a glutamine. One‐dimensional ^1^H‐NMR has been used to monitor Ca^2+^‐induced changes in the mutant proteins, and the spectral changes observed for each mutant have been compared to those for wild‐type CaM. In this way, the effect of each mutation on both the mutated site and the other Ca^2+^‐binding sites has been examined. The mutation of glutamate to glutamine at position 12 in any of the EF‐hand Ca^2+^‐binding loops greatly decreases the Ca^2+^‐binding affinity at that site, yet differs in the overall effects on Ca^2+^ binding depending on which of the four sites is mutated. When the mutation is in site I, there is only a small decrease in the apparent Ca^2+^‐binding affinity of site II, and vice versa. Mutation in either site III or IV results in a large decrease in the apparent Ca^2+^‐binding affinities of the partner C‐terminal site. In both the N‐ and C‐terminal domains, evidence for altered conformational effects in the partners of mutated sites is presented. In the C‐terminus, the conformational consequences of mutating site III or site IV are strikingly different.