The effect of Met-->Leu mutations on calmodulin's ability to activate cyclic nucleotide phosphodiesterase.
Calmodulin (CaM) has two hydrophobic surface patches that are particularly rich in Met residues, and these are the major contact areas where CaM interacts with its target enzymes. The amino acid Leu has been introduced by site-directed mutagenesis to replace all the Met residues in CaM. All nine individual Met-->Leu mutants of CaM as well as some double and quadruple mutants were expressed in Escherichia coli. All mutants could be purified by calcium-dependent hydrophobic affinity chromatography, indicating that they still expose their hydrophobic surfaces upon binding calcium. Each single Met-->Leu mutation in the C-terminal domain of the protein had little effect on its ability to activate phosphodiesterase (PDE), while a quadruple mutant with four C-terminal Leu residues instead of Met has a significantly lower affinity for PDE. The M36L mutant is a poor activator compared with the other three N-terminal single Met-->Leu mutants, which have a slightly lower affinity for PDE than wild-type CaM. The introduction of a positively charged Arg for Met-145 resulted in an almost complete loss of CaM's ability to activate PDE. Nuclear magnetic resonance spectroscopy was used to show that most CaM mutants retain their overall three-dimensional structure. Thus, the altered activation properties appear to arise from differences in the flexibility and polarizability of the Met and Leu sidechains, rather than from structural perturbations.
Zhang
M
M
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Li
M
M
Wang
J H
JH
Vogel
H J
HJ
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli; D007700:Kinetics; D007930:Leucine; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity
D002147:Calmodulin; D011994:Recombinant Proteins; D008715:Methionine; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D007930:Leucine