The effects of deletions in the central helix of calmodulin on enzyme activation and peptide binding (original) (raw)
1989, The Journal of Biological Chemistry
Using site-directed mutagenesis we have expressed in Escherichia coli three engineered calmodulins (CaM) containing deletions in the solvent-exposed region of the central helix. These are CaMA84, Glu-84 removed; CaMA83-84, Glu-83 and Glu-84 removed; and CaMA81-84, Ser-81 through Glu-84 removed. The abilities of these proteins to activate skeletal muscle myosin light chain kinase, plant NAD kinase, and bovine brain calcineurin activities were determined, as were their abilities to bind a synthetic peptide based on the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Similar results were obtained with all three deletion proteins. V , values for enzymes activated by the deletion proteins are all within 10-20% of those values obtained with bacterial control calmodulin. Relative to bacterial control values, changes in K,,, or K,j values associated with the deletions are all less than an order of magnitude: KaCt values for NAD kinase and myosin light chain kinase are increased 5-7-fold, K d values for binding of the synthetic peptide are increased 4-7-fold, and K,,, values for calcineurin are increased only 1-%fold. In assays of NAD kinase and myosin light chain kinase activation some differences between bovine calmodulin and bacterial control calmodulin were observed. With NAD kinase, K,,, values for the bacterial control protein are increased 4-fold relative to values for bovine calmodulin, and V , values are increased by 50%; with myosin light chain kinase, K,,, values are increased 2fold and V , values are decreased 10-15'70 relative to those values obtained with bovine calmodulin. These differences between bacterial control and bovine calmodulins probably can be attributed to known differences in postranslational processing of calmodulin in bacterial and eucaryotic cells. No differences between bovine and control calmodulins were observed in assays of calcineurin activation or peptide binding. Our observations indicate that contacts with the deleted The nucleotide sequence(s) reported in thispaper has been submitted 504 729.