Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

202 Berliner
Fig. 5. Low-temperature X-band EPR spectra of a 1 mM VO 2+ -α-LA complex,
pH 7.4 (10 mM HEPES buffer, T = 77 K). Experimental conditions were as follows:
frequency, 9.129 GHz; microwave power, 20 mW; modulation amplitude, 10 G; time
constant, 0. 128 s; scan time, 4 min; field set, 3400 G; scan range, 1600 G. Inset:
Upfield portion of the spectrum at fivefold higher gain. Reproduced with permission
from ref. 13.
4. Notes
1. When studying metal–protein complexes, it is important to try multifrequency
experiments. Although X-band (9.5 GHz) spectrometers are very common, the
other frequencies (S-band, Q-band, W-band) are available at some of the EPR
centers in the United States and various specialized labs around the world.
2. In order to verify that binding occurred at the principal binding site, the EPR
spectra must be measured in excess calcium in order to displace the paramagnetic
ion. One must still be cautious to check that secondary-site binding occurs
between the displaced paramagnetic ion and the protein (14).
3. More detailed spectral features of the electronic coordination to the cation are
more discernible in frozen solution. The most ideal situation would be single
crystals of metal-bound protein.
4. Spin-labeled proteins must be exhaustively dialyzed or chromatographed to
remove all remnants of free unreacted label. A second, more difficult to reconcile
problem is that where the protein is partially proteolyzed or denatured after labeling.
Occasionally, gel permeation or sophisticated HPLC methods might separate
away the approx 1–5% damaged protein which nonetheless contributes an
intense narrow three-line component to the spin-label spectrum.
5. In order to emphasize the covalently bound components of spin-labeled proteins,
it is often desirable to slow the motion of the overall protein or local (labeled)