CALCIUM-BINDING DECREASES THE STOKES RADIUS OF CALMODULIN AND MUTANTSR74A, R90A, AND R90G

Calmodulin (CaM) is an intracellular cooperative calcium-binding prote in essential for activating many diverse target proteins. Biophysical studies of the calcium-induced conformational changes of CaM disagree on the structure of the linker between domains and possible orientatio ns of the domains. Molecular dynamics studies have predicted that Ca(4 )(2+)CaM is in equilibrium between an extended and compact conformatio n and that Arg74 and Arg90 are critical to the compaction process. In this study gel permeation chromatography was used to resolve calcium-i nduced changes in the hydrated shape of CaM at pH 7.4 and 5.6. Results showed that mutation of Arg74 to Ala increases the R(s) as predicted; however, the average separation of domains in Ca-4(2+)-CaM was larger than predicted by molecular dynamics. Mutation of Arg90 to Ala or Gly affected the dimensions of apo-CaM more than those of Ca-4(2+)-CaM. C alcium binding to CaM and mutants (R74A-CaM, R90A-CaM, and R90G-CaM) l owered the Stokes radius (R(s)). Differences between R(s) values repor ted here and R(g) values determined by small-angle x-ray scattering st udies illustrate the importance of using multiple techniques to explor e the solution properties of a flexible protein such as CaM.