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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.

Publisher Summary This chapter discusses how synthetic oligonucleotides are used in studies of calmodulin and related calcium-binding proteins. The focus is on two specific applications of synthetic oligonucleotides: site-specific mutagenesis and detection of recombinant DNA vectors containing DNA inserts coding for calmodulin-like structures. The database of information on the phylogeny and ontogeny of calmodulin provides the necessary background for the initiation of studies utilizing a direct approach to structure and function that combines recombinant DNA, comparative enzymology, and protein chemistry techniques. The synthetic calmodulin gene was designed for mutagenesis by two approaches: cassette-based and M13-based mutagenesis. Mutagenesis is performed on a construction consisting of the synthetic calmodulin gene cloned in the EcoRI and BamHI sites of pUC8. Once the mutant calmodulin gene sequence is generated and verified, an expression vector containing the mutated gene sequences is constructed. Calmodulin mutant genes are expressed by cloning on the expression plasmid pKK223-3. The final purified product is characterized by a variety of analytical techniques including SDS-polyacrylamide gel electrophoresis, amino acid composition analyses of acid hydrolysates of the whole protein and selected peptide fractions, and limited amino acid sequence analyses.