Chemical shift assignments of calmodulin under standard conditions at neutral pH

Abstract

The Ca²⁺ sensor protein, calmodulin (CaM) is ubiquitously expressed in all cells where it binds to hundreds of different target proteins, including dozens of enzymes, receptors, ion channels and numerous Ca²⁺ transporters. The only published NMR chemical shift assignments for Ca²⁺-bound CaM (in the absence of a target) have been determined under acidic conditions: at pH 6.5/310 K (BMRB 6541) and pH 6.3/320 K (BMRB 547). However, some CaM/target complexes are not soluble under these conditions. Also, amide chemical shifts are very sensitive to pH and temperature, which can cause large baseline errors when using the existing chemical shift assignments of free CaM to calculate chemical shift perturbations caused by target binding at neutral pH and physiological temperature. We report complete NMR chemical shift assignments of Ca²⁺-saturated CaM under a set of standard conditions at neutral pH and 308 K that will enable more accurate chemical shift comparison between free CaM and CaM/target complexes (BMRB 51289).

Keywords: CaM; Calcium; Chemical shift perturbation; EF-hand; NMR.

Fig. 1

Two-dimensional NMR spectra of CaM under standard conditions at neutral pH. A ¹⁵N–¹H HSQC spectrum recorded at 600 MHz ¹H frequency was analyzed to determine backbone resonance assignments. The spectrally crowded regions are highlighted with black-dashed boxes (see inset). B Constant-time ¹³C–¹H HSQC spectrum was analyzed to determine aliphatic side chain resonance assignments. Representative resonance assignments are indicated by residue labels; complete assignments are available as BMRB accession no. 51289

Fig. 2

Secondary structure and RCI order parameters of CaM predicted from the assigned backbone chemical shifts. A Probability of secondary structural elements (cyan for helix and magenta for strand) and B RCI order parameter (RCI-S²) of CaM were predicted using TALOS + server (Shen et al. 2009). The wire diagram depicting the secondary structural elements (cylinder for helix and triangle for strand) was obtained from the CaM structure (PDB ID—2VAY (Halling et al. 2009))

Fig. 3

Amide chemical shift perturbation (CSP) for human CaM at neutral pH (BMRB 51289) compared to A frog CaM at pH 6.5 and 310 K (BMRB 6541) (Kainosho et al. 2006) and B drosophila CaM at pH 6.3 and 320 K (BMRB 547) (Ikura et al. 1990). CSP was calculated as: $CSP=\sqrt{{\left(\mathrm{\Delta },H^N\right)}^2+{\left(\mathrm{\Delta },N\right)}^2}$. ΔH^N and ΔN are the observed difference in the ¹H^N and ¹⁵N chemical shifts, respectively between CaM at pH 7.0 (BMRB 51289) and CaM at either pH 6.5 (BMRB 6541) or pH 6.3 (BMRB 547). C Side-chain methyl chemical shift perturbation was calculated as: $CSP=\sqrt{{(\mathrm{\Delta }H)}^2+{(\mathrm{\Delta }C)}^2}$. ∆H and ∆C are the observed difference in ¹H and ¹³C methyl chemical shifts between 51,289 and 6541. CSP values are mapped on to the CaM structure (PDB ID: 2VAY (Halling et al. 2009))