Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

FTIR Spectroscopy of Calcium-Binding Proteins 63
Fig. 2. (A) IR spectra of apo-calmodulin (c approx 11 mg/mL) in D 2O-buffer (100 mM
Na-cacodylate, pH 7.0) after complete H/D exchange (solid line), together with the buffer
spectrum (dashed line). For complete exchange of all amide protons with deuterons, the
lyophylized protein was dissolved in D 2O-buffer and left overnight at room temperature.
To eliminate the possibility of significant Ca 2+ -leaking from the infrared cell, the apo protein
sample was placed in the CaF 2 cell immediately before the measurement, which was
completed within less than 10 min. Infrared spectra were recorded on a Bruker IFS-66
FTIR spectrometer equipped with a DTGS detector. For each sample, 128 interferograms
were co-addded and Fourier-transformed applying a Happ-Genzel apodization function to
generate a spectrum with a nominal resolution of 4 cm –1 . (B) IR spectrum of the fully
exchanged apo-form after subtraction of the buffer spectrum (solid line). The dashed line
shows a correponding difference spectrum of only partly exchanged apo-CaM recorded
15 min after dissolution of the lyophilized protein in D 2O.
3.2.2. Residual Water Vapor
It is almost impossible to remove all water vapor by purging of the spectrometer.
In addition, the level always changes when the sample chamber is opened.
It is therefore convenient to record spectra at low, but well-matched, levels of