SMALL-ANGLE X-RAY-SCATTERING STUDIES OF CALMODULIN MUTANTS WITH DELETIONS IN THE LINKER REGION OF THE CENTRAL HELIX INDICATE THAT THE LINKER REGION RETAINS A PREDOMINANTLY ALPHA-HELICAL CONFORMATION

Two mutant forms of calmodulin were examined by small-angle X-ray scattering in solution and compared with the wild-type protein. Each mutant has deletions in the linker region of the central helix: one lacks residues Glu-83 and Glu-84 (Des2) and the other lacks residues Ser-81 through Glu-84 (Des4). The deletions change both the radii of gyration and the maximum dimensions of the molecules. In the presence of Ca2+, the observed radii of gyration are 22.4 angstrom for wild-type bacterially expressed calmodulin, 19.5 angstrom for Des2 calmodulin, and 20.3 angstrom for Des4 calmodulin. A reduction in the radius of gyration by 1-2 angstrom on removal of calcium, previously observed in the native protein, was also found in the wild type and the Des4 mutant; however, no significant size change was observed in the Des2 mutant. The large calcium-dependent conformational change in calmodulin induced by the binding of melittin [Kataoka, M., Head, J. F., Seaton, B. A., & Engelman, D. M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6944-6948] was observed in all the bacterially expressed proteins. Each protein appears to undergo a transition from a dumbbell shape to a more globular conformation on binding melittin in the presence of calcium, although quantitatively the changes in the wild-type and Des4 proteins greatly exceed those in Des2. Modeling shows that the structural properties of the deletion mutants are well described by modifications of an alpha-helix in the central linker region of the molecule. Thus, the structure of the linker region is stable enough to maintain the average orientation and separation of the lobes yet flexible enough to permit the lobes to approach each other upon binding a peptide.