Stopped-flow studies of calcium dissociation from calcium-binding-site mutants of Drosophila melanogaster calmodulin.
The kinetics of calcium dissociation from two groups of site-specific mutants of calmodulin from Drosophila melanogaster have been studied by stopped-flow kinetic methods, using the fluorescent calcium chelator 8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6- methoxyquinoline-N,N,N',N'-tetraacetic acid (Quin 2). The BQ series of mutants consists of four proteins in which one of the four bidentate glutamate residues (Glu12 of each of the four calcium binding loops) has been replaced by glutamine. In the BK series of mutants, the corresponding glutamate has been replaced by lysine. Calcium-dissociation kinetics of proteins with a mutation in site I or II (N-terminal domain) are consistent with a model in which the mutation weakens binding at the non-mutated N-terminal partner site and has a small, but significant, effect on the kinetic properties of sites III and IV (C-terminal domain). The proteins with a mutation in site III or IV show a large effect, with decreased Ca2+ dissociation rate from the unmodified N-terminal Ca(2+)-binding sites I and II. A structural interpretation is proposed, based on enhanced interactions between the domains when the affinity of individual sites have been dramatically reduced by mutation. This effect is greatest for the mutations in the C-terminal domain, which appear to destroy the co-operativity of Ca2+ binding at sites III and IV. The results show that site-specific mutation can have surprisingly far-ranging effects on kinetic properties of calmodulin. The kinetic analysis also shows that studies of specifically engineered mutants may in principle help to unmask the values of intrinsic rate constants for the wild-type protein which are not normally observable in the process of Ca2+ dissociation.
Martin
S R
SR
Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London, England.
Maune
J F
JF
Beckingham
K
K
Bayley
P M
PM
D000595:Amino Acid Sequence; D000634:Aminoquinolines; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism; D002413:Cations, Divalent; D003198:Computer Simulation; D004331:Drosophila melanogaster / Q000378:metabolism*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation
D000634:Aminoquinolines; D002147:Calmodulin; D002413:Cations, Divalent; D005456:Fluorescent Dyes; C035739:Quin2; D002118:Calcium