Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Cadmium-113 and Lead-207 NMR Studies 209
Fig. 2. 207 Pb NMR spectra of intact CaM and its C-terminal domain fragment, TR 2C
(residues 78–148). Four signals are observed in the spectra; the two sharp signals can
be assigned to the C lobe by lining up with the two peaks for the half-molecule of
CaM, whereas the two broad peaks can be attributed to the other two calcium-binding
sites in the N lobe (8).
additional broad peaks not present in the room temperature spectra of TnC,
which correspond to the N-terminal domain-binding sites, can be observed.
However, even at low temperatures, additional resonances for the two weak
binding sites of CaM are not present in the spectra (see bottom panel of Fig. 3).
In this case, it is likely that the C-domain of the protein is static, whereas the
N-domain is rapidly changing conformation. If the N-terminal 113 Cd 2+ ions
have different shifts in these different conformations, the spectra obtained at
different exchange rates can be simulated (see Fig. 4). The spectra obtained for
CaM at room temperature match the spectrum calculated for an exchange rate
(k exch) of 10 4 s –1 (2,3). Often, it is thought that the exchange process responsible
for the broadening involves free Cd 2+ in solution; however, in the case of
CaM, when a fifth equivalent is added, a free Cd 2+ signal can be observed,
which is in slow exchange (data not shown). Hence, a conformational exchange
process must be occurring in the N-terminal domain, as indicated in Fig. 4.
When a peptide binds to CaM, the N-domain adopts one unique conformation
and with the exchange process eliminated, four peaks appear in the spectra for
the four calcium-binding sites of CaM (see top panel of Fig. 3) (13).