1H, 15N, and 13C chemical shift assignments of calcium-binding protein 1 with Ca2+ bound at EF1, EF3 and EF4

Abstract

Calcium-binding protein 1 (CaBP1) regulates inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) and a variety of voltage-gated Ca(2+) channels in the brain. We report complete NMR chemical shift assignments of the Ca(2+)-saturated form of CaBP1 with Ca(2+) bound at EF1, EF3 and EF4 (residues 1-167, BMRB no. 16862).

Fig. 1

Two-dimensional ¹⁵N-¹H HSQC spectrum of Ca²⁺-saturated CaBP1 recorded at 800-MHz ¹H frequency. The protein sample (1 mM) was uniformly labeled with nitrogen-15 and was dissolved in 0.3 ml of a 90% H₂O/10% [²H] H₂O solution containing 10 mM [²H₁₁] Tris (pH 7.4), 1 mM [²H₁₀] dithiothreitol, and 5 mM CaCl₂. Under these conditions, CaBP1 contains Ca²⁺ bound at EF1, EF3 and EF4 (Wingard et al. 2005)

Fig. 2

Amide chemical shift differences between Ca²⁺-free (Li et al. 2009) and Ca²⁺-saturated CaBP1 (this study). a shows Δδ_N(ppm) = δ_N(Ca²⁺-free)—δ_N(Ca²⁺-bound) and b shows Δδ_H(ppm) = δ_H(Ca²⁺-free)—δ_H (Ca²⁺-bound). Residues in the EF-hand binding loops display the largest chemical shift differences and are highlighted