Studies of the roles of the individual calcium binding sites of Drosophila calmodulin in conformational change and target interaction

[摘要] The binding of calcium (Ca$sp{2+}$) to the four EF-hands of the ubiquitous, eukaryotic protein, calmodulin (CaM) generates the biologically active conformation of the protein. The roles of the four individual Ca$sp{2+}$-binding sites of Drosophila CaM had previously been examined using mutants in which the conserved glutamic acid in each site has been mutated to glutamine. In this thesis, the molecular mechanism of activation of target enzymes by CaM has been studied by the use of these mutants. Our principal finding is that most of these mutant proteins form complexes with target peptides which are conformationally distinct from the wild-type complex. However, mutation of the four sites differ significantly in the severity of their effects. This demonstrates the unique contributions of the individual Ca$sp{2+}$-binding events to the conformational changes required for target interaction. Studies of the peptide pattern generated by different site-specific proteases have indicated that loss of Ca$sp{2+}$-binding leads to conformational differences between the proteins. Multiple binding site mutants of CaM, including mutants in which sites 1+2, 1+3, 2+4, 3+4, or all four sites have been mutated have also been examined. Ca$sp{2+}$-induced changes have been studied by UV difference spectroscopy and circular dichroism (CD) spectra in the near and far UV regions. Several lines of evidence indicate that the environment of the single tyrosine residue (Tyr-138 in the fourth Ca$sp{2+}$-binding loop) is influenced by both N- and C-terminal mutations implying the existence of interdomain interaction. To examine N-terminal conformation changes on Ca$sp{2+}$-binding to the C-terminus, the mutant F65W was created with a unique tryptophan (Trp) residue in the second Ca$sp{2+}$-binding site. This Trp mutation has been combined with the Q mutants in the first, third and fourth Ca$sp{2+}$-binding sites and Ca$sp{2+}$-induced changes in the fluorescence of the Trp residue have been monitored. The activation pattern of one CaM-regulated enzyme, skeletal muscle myosin light chain kinase has indicated that for these mutant proteins the presence of the target protein could not compensate for lack of Ca$sp{2+}$-binding and an active conformation of the protein could not be generated.

[发布机构] Rice University