Protein surface charges and Ca2+ binding to individual sites in calbindin D9k: stopped-flow studies.
The kinetics of calcium dissociation from two groups of site-specific mutants of calbindin D9k--a protein in the calmodulin superfamily with two Ca2+ sites and a tertiary structure closely similar to that of the globular domains of troponin C and calmodulin--have been studied by stopped-flow kinetic methods, using the fluorescent calcium chelator Quin 2, and by 43Ca NMR methods. The first group of mutants comprises all possible single, double, and triple neutralizations of three particular carboxylate groups (Glu-17, Asp-19, and Glu-26) that are located on the surface of the protein. These carboxylates are close to the two EF-hand calcium binding sites, but are not directly liganded to the Ca2+ ions. Conservative modification of these negative carboxylate side chains by conversion to the corresponding amides results in a marked reduction in the Ca2+ binding constants for both sites, as recently reported [Linse et al. (1988) Nature 335, 651-652]. The stopped-flow kinetic results show that this reduction in Ca2+ affinity derives primarily from a reduction in the Ca2+ association rate constant, kon. The estimated maximum value of the association rate constant (kon(max) for Ca2+ binding to the wild-type protein is ca. 10(9) M-1 s-1. In contrast, for the mutant protein with three charges neutralized the maximum association rate constant is estimated to be only 2 X 10(7) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Martin
S R
SR
Division of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London, England.
Linse
S
S
Johansson
C
C
Bayley
P M
PM
Forsén
S
S
D001665:Binding Sites; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D064030:S100 Calcium Binding Protein G / Q000378:metabolism*; D013499:Surface Properties
D064026:Calbindins; D064030:S100 Calcium Binding Protein G; D002118:Calcium