Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Cadmium-113 and Lead-207 NMR Studies 207
A 2-mL protein sample is prepared in 90% H 2O, 10% D 2O, 100 mM KCl, and
the pH is adjusted to 7.5 (or desired pH) with small amounts of 0.5 M KOD or
0.5 M DCl (see Note 5). Ideally, the concentration of the protein sample (determined
spectrophotometrically) should be at least 1 mM for obtaining a reasonable
signal-to-noise ratio.
3.2. Spectral Acquisition
Typical 113Cd NMR spectra (88.75 MHz) are acquired locked with a pulse
length of 45–60°, sweep widths of 30 kHz, repetition rates of 0.50–0.60 s, and
4–8 k data points. When acquired in this fashion, the spectra are not usually
fully relaxed and quantification by integration of the area under the peak is not
reliable. In order to be able to make a relative comparison of the amount of
Cd2+ under each peak, the 113Cd nucleus must be fully relaxed; to determine an
appropriate delay time between pulses, the delay time is increased until the
amplitude of the signal ceases to increase. Each free induction decay (FID) is
generally zero filled once and processed with an exponential multiplication
function typically resulting in a line broadening of 30 Hz. The external chemical
shift reference is either 100 mM 113Cd(ClO4) 2 or 113CdSO4 in D2O. 207Pb NMR (83.45 MHz) acquisition parameters include 60–80° pulses,
sweep widths of 100 kHz, 0.40–0.55 s between pulses, and 16 k data points.
Large sweep widths are employed because of the wide chemical shift window
of 207Pb, but unequal excitation of the signal could result (8). During processing,
spectra are usually left-shifted, giving an effective dead time of 50–70 µs,
zero filled once, with an exponential line broadening of 300–500 Hz.
207Pb(NO3) 2 in D2O can be used as the external chemical shift reference.
The sample is inserted into the instrument and a number of scans are taken.
The number of scans required depends upon the concentration of the sample,
the desired noise level of the spectra, and the time allotted for the experiment.
For 113Cd NMR, this is typically 50,000 scans for a 1-mM protein solution,
whereas 100,000 scans are required for 207Pb NMR spectra at the same concentration,
taking a full day for data acquisition. During a metal-ion titration, the
pH of the sample is checked after each addition of metal solution before the
spectra are obtained. Figure 1 shows a 113Cd NMR spectrum obtained for
calbindin D9K (ICaBP, intestinal calcium-binding protein), and, for comparison,
typical chemical shifts for several other calcium-binding proteins including
calmodulin (CaM), troponin C (TnC), parvalbumin, and lactalbumin (12).
Figure 2 shows a 207Pb NMR spectrum of CaM. Two narrow peaks present in
the spectrum, correspond to slow exchange binding with two calcium-binding
sites (III and IV, assigned by proteolytic fragments, see Subheading 3.4.),
whereas the other broad resonances are attributed to the other two binding sites
(I and II) of CaM (8). During titration experiments with CaM, sites III and IV