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).

Chemical shift assignments of calmodulin bound to the GluN1 C0 domain (residues 841–865) of the NMDA receptor

“…2 ). The CaM/GluN1 C0 secondary structure is similar to that reported previously for free CaM (Bej and Ames 2022b ), and is depicted by cylinders and triangles in Fig. 2 A.…”

“…A Backbone amide CSP was calculated as: . ΔH N and ΔN are the observed difference in the 1 H N and 15 N chemical shifts, respectively between peptide-bound and unbound Ca 2+ -saturated CaM (Bej and Ames 2022b ). B Side-chain methyl CSP was calculated as: .…”

Chemical shift assignments of calmodulin bound to a C-terminal site (residues 1120–1147) in the β-subunit of a retinal cyclic nucleotide-gated channel (CNGB1)

“…Three residues in the second EF-hand of CaM (A74, R75, and K76) could not be assigned, because their HSQC peaks could not be detected. These same resonances are exchange broadened in CaM bound to the CNGB1 CaM1 peptide (Bej and Ames 2022a ) and the α-subunit of the retinal cyclic nucleotide-gated channel (CNGA2) (Contessa et al 2005 ), but are not exchange broadened in free CaM (Bej and Ames 2022b ; Kainosho et al 2006 ). The chemical shift assignments ( 1 H, 15 N, 13 C) for CaM/CaM2 have been deposited in the BioMagResBank ( http://www.bmrb.wisc.edu ) under accession number 51447.…”

“…3 Chemical shift perturbation (CSP) versus residue number for CaM/CaM2. CSP was calculated as: , where ΔH N and ΔN are the difference in the 1 H N and 15 N chemical shifts, respectively for CaM/CaM2 versus free CaM in the absence of CaM2 (Bej and Ames 2022b ). CSP values are superimposed on the CaM crystal structure (PDB ID: 2VAY (Halling et al 2009 )) …”