CaBP Data Library General Information

Information in the EF-Hand Calcium-Binding Proteins Database from Reference 260

Protein
 Species
 Information
 Comment
 Submitted By
 Validated By
calbindin D9k
 Bos taurus
(cow or bovine)
 Report of an engineered mutant with following mutations:
  • E60D
Melanie R. Nelson
The Scripps Research Institute, Department of Molecular Biology
mnelson@scripps.edu
 Melanie R. Nelson
mnelson@scripps.edu
calbindin D9k
mutant:
  • E60D
 Bos taurus
(cow or bovine)
 Ca binding constants measured by competitive chelator:
  • K1 = 7.94E06
  • K2 = 2.51E08
  • Temperature: 25
  • pH: 7.5
  • Salt concentration: no salt added
  • Buffer:
    • 0.002 M Tris-HCl
 The chelator used was 5, 5'-Br2BAPTA
 Melanie R. Nelson
The Scripps Research Institute, Department of Molecular Biology
mnelson@scripps.edu
 Melanie R. Nelson
mnelson@scripps.edu
calbindin D9k
mutant:
  • E60D
 Bos taurus
(cow or bovine)
 Ca binding constants measured by competitive chelator:
  • K1 = 251000
  • K2 = 3.16E06
  • Temperature: 25
  • pH: 7.5
  • Salt concentration: 150 mM KCl
  • Buffer:
    • 0.002 M Tris-HCl
 The chelator used was 5, 5'-Br2BAPTA
 Melanie R. Nelson
The Scripps Research Institute, Department of Molecular Biology
mnelson@scripps.edu
 Melanie R. Nelson
mnelson@scripps.edu
calbindin D9k
mutant:
  • E60D
 Bos taurus
(cow or bovine)
 This protein binds 2 equivalents of Ca ion
Melanie R. Nelson
The Scripps Research Institute, Department of Molecular Biology
mnelson@scripps.edu
 Melanie R. Nelson
mnelson@scripps.edu
calbindin D9k
mutant:
  • E60D
 Bos taurus
(cow or bovine)
 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
Melanie R. Nelson
The Scripps Research Institute, Department of Molecular Biology
mnelson@scripps.edu
 Melanie R. Nelson
mnelson@scripps.edu