Calbindin D9k Mutant: E60D
Engineered mutant of calbindin D9k, from Bos taurus (InfoCard)
General Effects of the Mutation:
- In this mutant, calcium affinity is decreased by approximately 38-fold. This reduction in affinity is almost completely due to the first binding step. The cooperativity of calcium binding is significantly higher in this mutant than in wildtype calbindin. The loss in affinity is thought to be due to the shorter length of the asp sidechain. The shorter length weakens the hydrogen bond between the sidechain and a water molecule which is a calcium ligand in site I. The reason for the increase cooperativity is less clear. The x-ray structure of the mutant and analysis of chemical shift changes in the mutant indicate that the structural changes in the calcium-loaded state are minor, mostly confined to residues E60D, Q22,and D19. Chemical shift analysis of the apo state also indicates only minor changes, but over a larger region of the protein (InfoCard)
Physical Properties of the Mutant:
Ca Binding Constants (low salt:) :
K1 = 7.94 x 106; K2 = 2.51 x 108
(measured by: competitive chelator pH: 7.5 temperature: 25 degrees Celsius buffer: 0.002 M Tris-HCl )
(InfoCard)
Ca Binding Constants (high salt:) :
K1 = 2.51 x 105; K2 = 3.16 x 106
(measured by: competitive chelator pH: 7.5 temperature: 25 degrees Celsius salt: 150 mM KCl buffer: 0.002 M Tris-HCl )
(InfoCard)