AN INTERDISCIPLINARY APPROACH TO THE MOLECULAR MECHANISMS OF CALMODULIN ACTION: COMPARATIVE BIOCHEMISTRY, SITE-SPECIFIC MUTAGENESIS, AND PROTEIN ENGINEERING

Publisher Summary This chapter presents an interdisciplinary approach to the molecular mechanisms of calmodulin action. The VU-1 calmodulin has an amino acid sequence that is a hybrid of vertebrate and plant calmodulins. The bacterially expressed protein lacks two of the post-translational modifications common to vertebrate and some other calmodulins. These include the acetylation of the amino terminus and the trimethylation of lysine-115. Apart from the lack of acetylation, the changes in the VU-1 calmodulin structure have been observed in the naturally occurring calmodulins. The study of calmodulin-activated proceses by protein engineering requires structural information about specific target enzymes. The detailed studies have focused on the calmodulin dependent enzyme Myosin Light Chain Kinase (MLCK). The amino acid sequences of potential calmodulin binding sites have been elucidated for skeletal and smooth muscle MLCK. A new calmodulin expression vector that allows for mutagenesis, amplification, characterization and expression of mutant calmodulin genes in E. coli has been constructed. The new expression vector, pVUCH-1, is a hybrid of pVUC-1 and pUC8. The orientation of the gene relative to the tac promoter elements within the plasmid is the same as that found in pVUC-1.