Calcium binding decreases the stokes radius of calmodulin and mutants R74A, R90A, and R90G.
Calmodulin (CaM) is an intracellular cooperative calcium-binding protein 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 orientations of the domains. Molecular dynamics studies have predicted that Ca4(2+)CaM is in equilibrium between an extended and compact conformation and that Arg74 and Arg90 are critical to the compaction process. In this study gel permeation chromatography was used to resolve calcium-induced changes in the hydrated shape of CaM at pH 7.4 and 5.6. Results showed that mutation of Arg 74 to Ala increases the R(s) as predicted; however, the average separation of domains in Ca4(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 Ca4(2+)-CaM. Calcium binding to CaM and mutants (R74A-CaM, R90A-CaM, and R90G-CaM) lowered the Stokes radius (R(s)). Differences between R(s) values reported here and Rg values determined by small-angle x-ray scattering studies illustrate the importance of using multiple techniques to explore the solution properties of a flexible protein such as CaM.
Sorensen
B R
BR
Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242-1109, USA.
Shea
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D017931:DNA Primers; D006863:Hydrogen-Ion Concentration; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D002147:Calmodulin; D017931:DNA Primers; D011994:Recombinant Proteins; D002118:Calcium