Electrostatic contributions to the binding of Ca2+ in calbindin D9k.
A set of accurate experimental data is provided for Ca2+ ion binding to calbindin D9k, a protein in the calmodulin superfamily of intracellular regulatory proteins. The study comprises both the role of protein surface charges and the effects of added electrolyte. The two macroscopic Ca2(+)-binding constants K1 and K2 are determined for the wild-type and eight mutant calbindins in 0, 0.05, 0.10, and 0.15 M KCl from titrations in the presence of Quin 2 or 5,5'-Br2BAPTA. The mutations involve replacement of surface carboxylates (of Glu17, Asp19, Glu26, and Glu60) with the corresponding amides. It is found that K1K2 may decrease by a factor of up to 2.5 x 10(5) (triple mutant in 0.15 M KCl as compared to the wild-type protein in 0 M KCl). Ca2(+)-binding constants of the individual Ca2+ sites (microscopic binding constants) have also been determined. The positive cooperativity of Ca2+ binding, previously observed at low salt concentration [Linse et al. (1987) Biochemistry 26, 6723-6735], is also present at physiological ionic strength and amounts to 5 kJ.mol-1 at 0.15 M KCl. The electrolyte concentration and some of the mutations are found to affect the cooperativity. 39K NMR studies show that K+ binds weakly to calbindin. Two-dimensional 1H NMR studies show, however, that potassium binding does not change the protein conformation, and the large effect of KCl on the Ca2+ affinity is thus of unspecific nature. Two-dimensional 1H NMR has also been used to assess the structural consequences of the mutations through assignments of the backbone NH and C alpha H resonances of six mutants.(ABSTRACT TRUNCATED AT 250 WORDS)
Linse
S
S
Chemical Centre, Lund University, Sweden.
Johansson
C
C
Brodin
P
P
Grundström
T
T
Drakenberg
T
T
Forsén
S
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D004563:Electrochemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D008958:Models, Molecular; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D011487:Protein Conformation; D064030:S100 Calcium Binding Protein G / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D064026:Calbindins; D064030:S100 Calcium Binding Protein G; D002104:Cadmium; D002118:Calcium