Circular dichroism studies on calcium binding to two series of Ca2+ binding site mutants of Drosophila melanogaster calmodulin.
The Ca(2+)-induced structural changes in mutant calmodulins from Drosophila melanogaster have been studied by circular dichroism. The proteins comprise eight site-specific mutants, in which a bidentate glutamic acid (at position 12 in each Ca2+ binding loop) is replaced with either glutamine (BQ series) or lysine (BK series). Previous studies of these proteins indicate that Ca2+ binding at the mutated site is effectively eliminated by each of these substitutions, with additional effects at nonmutated sites. Circular dichroism has now been used to assess Ca(2+)-induced changes in secondary and tertiary structure in these proteins. In the absence of Ca2+, the helical content of these mutant calmodulins is close to that of the wild-type protein. In excess Ca2+, calmodulins with a mutation in the N-terminal sites show Ca(2+)-induced increases in helicity (CD at 222 nm) that are similar to those of the wild-type protein. In contrast, much less additional helix is induced by Ca2+ in calmodulins with mutations in the C-terminal sites, with the two mutations to site IV showing a particularly poor response. Ca(2+)-induced changes to the environment of the single tyrosine of Drosophila calmodulin (Tyr-138 in site IV of the C-terminal domain) have been monitored via CD at 280 nm. The signal from this residue is significantly altered in the Ca(2+)-free form of almost all these mutants, including those in the N-terminal domain. This indicates significant interaction between the N- and C-terminal domains of these mutants.(ABSTRACT TRUNCATED AT 250 WORDS)
Maune
J F
JF
Department of Biochemistry and Cell Biology 2, Rice University, Houston, Texas 77251.
Beckingham
K
K
Martin
S R
SR
Bayley
P M
PM
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004331:Drosophila melanogaster / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation
D002147:Calmodulin; D010649:Phenylalanine; D002118:Calcium