Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Cadmium-113 and Lead-207 NMR Studies 213
Fig. 6. Relationship of the CaM-bound 113 Cd and 207 Pb linewidths (∆v 1/2) with
the square of the magnetic field strength (B o). � and � from 207 Pb CaM peaks at δ = 983
ppm and δ = 965 ppm, respectively. � from 113 Cd CaM signal at δ = –114.5 ppm.
crystal structures of some calcium-binding proteins with Cd 2+ are identical to
those with Ca 2+ , lending credence to the substitution method (18).
4. Notes
1. It is not always trivial to completely remove EDTA or EGTA once they have been
introduced into the protein preparation so treatment with Chelex is preferred
whenever possible. Peaks from remaining EDTA or EGTA contamination can
sometimes be detected in the NMR spectra.
2. For lower-affinity Ca2+ -binding sites, chemical exchange rates between free and
bound Cd2+ may cause signals to be too broad for detection. On the other hand,
broad resonances could result from conversion between two or more protein conformations
(see Fig. 4). These two possibilities must be considered; temperature
variation experiments are very useful in this respect. The chemical exchange rate
for 207Pb2+ bound to calmodulin is also very temperature sensitive, with peaks
disappearing above or below ambient temperature (8).
3. NMR sensitive nuclei with a large electron cloud such as 113Cd or 207Pb often
relax through a mechanism known as chemical shift anisotrophy (CSA). For protein-bound
nuclei, this effect becomes rather substantial and should be considered
when selecting an NMR spectrometer, which will give optimal results.
Figure 6 shows the consequence of CSA on 113Cd and 207Pb NMR spectra of
CaM, recorded on 100 MHz, 300 MHz, 400 MHz, and 500 MHz instruments.