Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

210 Clarke and Vogel
Fig. 3. 113 Cd NMR spectra of CaM and a 1:1 complex with MLCK (myosin lightchain
kinase) peptide. Only two signals are present for cadmium-saturated CaM, corresponding
to the two C-terminal sites, whereas the two signals for the N-terminal
sites are broadened beyond detection. Upon addition of peptide, four signals at different
chemical shifts appear, matching each of the four sites. Binding of the peptide
abolishes the conformational exchange process in the N-terminal domain of CaM (see
Fig. 4) (13).
3.4. Using Proteolytic Fragments to Assign Peaks to Binding Sites
Individual domains from many calcium-binding proteins can be isolated by
limited proteolytic degradation with various enzymes (see Note 6) (11). Structurally
intact binding sites produce spectra with very similar chemical shifts as
the intact sites in the whole protein (13,14). Ideally, each metal-ion binding site
of the protein would be isolated for efficient identification of each resonance.
However, it is sometimes possible to assign resonances with fragments containing
more than one binding site. For example, calmodulin can be digested
with either thrombin or trypsin to produce different fragments containing varying
numbers of binding sites (15). NMR samples containing the proteolytic
fragments of the protein are prepared similarly to those for the intact protein
and experimental parameters also remain the same.
An example is shown in the 207 Pb NMR spectra in Fig. 2, where the two
narrow signals for CaM clearly arise from the C lobe (TR 2C, residues 78–142)
of the protein, and by elimination, the other two signals are from the N-lobe of
the protein (TR 1C, residues 1–77). Slight differences are sometimes found
between the chemical shifts for the fragments and whole protein. These may be
a result of slight perturbations in the Ca 2+ -binding loops of the binding site,
caused by proteolytic cleavage.