The overall objective of the proposed research is to utilize recombinant DNA methodology to produce calmodulins (CaM) that have specific alterations introduced into the primary amino acid sequence. The alterations are introduced at the nucleic acid level by combining selected portions of a full-length chicken CaM cDNA (pCaM 16) or eel CaM cDNA (pCM 116) with portions of a calmodulin-like genomic clone (CM-1). CM-1 contains 19 amino acid substitutions relative to CaM of which 11 are non-conservative. These changes include the appearance of a cysteine residue in Ca++ binding domains I and IV at positions 26 and 130, respectively. The recombinant molecules are introduced into a bacterial expression plasmid containing both trp and lac promoters and introduced into bacteria. The bacterially synthesized proteins are isolated by phenothiazine chromatography and used to evaluate the role of specific peptide domains in a variety of physicochemical properties of CaM. These properties will include Ca++ binding and Ca++ induced conformational changes, drug binding and the ability to bind to and activate two CaM dependent enzymes, cyclic nucleotide phosphodiesterase (PDE) and myosin light chain kinase (MLCK). Molecules will be constructed that contain the following alterations in the four Ca++ binding domains: 1) domains I, II and IV; 2) domain I; 3) domain II; 4) domains II and IV. In this manner it is hoped to gain insight into which of the Ca++ binding subdomains of CaM are involved in regulating many of the properties and biological roles attributed to this multifunctional Ca++ binding protein. The CM-1 clone hybridizes to a mRNA present in striated muscles of the chicken that is distinct from CaM mRNA. These data suggest the presence of a novel CaM-like molecule in muscle tissues. Therefore we have derived a strategy to demonstrate the presence of this protein, localize it to structural portions of the muscle and study its development during muscle differentiation compared to the development of CaM. These data would be the first to show the presence of a CaM-like molecule in vertebrate tissue and lead us to speculate that a """"""""processed"""""""" gene might be expressed in a tissue specific manner.
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