Sequence-Specific NMR Assignments for Calcium-Binding Proteins

Assignments for Calbindin D_9k

• Wildtype calbindin (P43M and P43G are included as wildtype, because these are the standard background mutations for all other mutations studied)
  • Apo P43M, pH = 5.3, T = 300 K
  • Apo P43M, pH = 6.01, T = 300 K
  • Calcium-loaded P43M, pH = 6.1, T = 300 K
  • Calcium-loaded P43M, pH = 6.02, T = 300 K
  • Calcium-loaded P43G
  • Calcium-loaded P43G (¹³C assignments), pH = 6.0, T = 300 K
• Mutant calbindins Scripps and Lund only
  • E27Q: Mutation of the conserved bidentate Glu in binding loop 1 completely destroys calcium binding in this loop. Assignments are very similar to wildtype (P43M). Most signficant differences are confined to binding loop 1.
    • Apo E27Q
    • E27Q with one calcium ion bound (to site II)
    • E27Q in the presence of excess calcium. Site I does not seem to bind calcium, even in the presence of more than 14 equivalents of calcium.
  • F36G: This mutation was designed to test a hypothesis about the reason for differences in conformation of the apo states of calbindin and calmodulin, and to possibly repack the apo state of calbindin
    • Apo F36G, pH = 5.99, T = 300 K
    • Calcium-loaded F36G, pH = 6.05, T = 300K
  • I73V: This mutation was designed to examine the role of I73 in the hydrophobic packing of calbindin. In the calcium-loaded structure of wildtype calbindin, the methyl protons pack under the ring of F36, shifting them to an unusually low frequency. The sidechain of valine seems to be too short to make this interaction with the aromatic ring of F36, for its sidechain protons resonate at normal frequencies.
    • Apo I73V, pH = 6.02, T = 300 K
    • Calcium-loaded I73V, pH = 5.3, T = 300 K

Assignments for Calcyclin

• apo calcyclin

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