title: Regulation of scallop myosin by mutant regulatory light chains.
abstract: Scallop adductor myosin is regulated by its subunits; the regulatory light chain (R-LC) and essential light chain (E-LC). Myosin light chains suppress muscle activity in the absence of calcium and are responsible for relaxation. The binding of Ca2+ to the myosin triggers contraction by releasing the inhibition imposed on myosin by the light chains. To map the functional domains of the R-LC, we have carried out mutagenesis followed by bacterial expression. Both wild-type and mutant proteins were hybridized to scallop myosin heavy chain/E-LC to map the regions of the light chain that are responsible for the binding to the myosin heavy chain/E-LC, for restoring the specific calcium-binding site, and controlling the myosin ATPase activity. The R-LC is expressed in Escherichia coli using the pKK223-3 (Pharmacia) expression vector and has been purified to greater than 90% purity. E. coli-expressed wild-type R-LC differs from the native R-LC by having the initiating methionine residue and an unblocked NH2 terminus. The wild-type R-LC restores Ca2+ binding and Ca2+ sensitivity when hybridized to scallop myosin. A point mutation of the sixth Ca2(+)-liganding position of domain I (Asp39----Ala39) results in a R-LC that binds more weakly to the heavy chain/E-LC and restores the specific Ca2(+)-binding site but not regulation of the actin-activated Mg2+ ATPase. A second mutation was produced by substituting the last 11 residues of the COOH terminus with 15 different residues. This mutant restores the specific Ca2(+)-binding site, but does not restore Ca2+ regulation to the actin-activated ATPase activity. Several other point mutations do not alter light chain function. The experiments directly establish that the divalent cation-binding site of domain I is functionally distinct from the specific Ca2(+)-binding site. The results indicate that an intact domain I and the COOH terminus are required to suppress the myosin ATPase activity. The fact that the domain I mutation and the COOH-terminal mutation disrupt regulation but do not affect Ca2(+)-binding indicates that these two aspects of regulation are separable and, therefore, the R-LC has distinct functional regions.
authors: Department of Biology, Brandeis University, Waltham, MA 02254.
authors: E B
authors: EB
authors: Goodwin
authors: L A
authors: LA
authors: Leinwand
authors: A G
authors: AG
authors: Szent-Györgyi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008974:Mollusca; D009132:Muscles / Q000378:metabolism / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D015183:Restriction Mapping
chemical_list: D009218:Myosins; D002118:Calcium