Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

FTIR Spectroscopy of Calcium-Binding Proteins 65
tein measurements, because the narrow water vapor bands overlap with the conformation-sensitive
amide I/II bands. For aqueous solutions, subtracting two
buffer spectra from the same cell collected at different purge levels should generate
the correct water vapor spectrum. Any over- or undersubtraction of water
vapor can best be visualized by calculating the second derivatives of the spectra,
which enhances narrow bands in particular (5). The subtraction factor must be
varied until the second-derivative spectrum is featureless in the range 1750–
1850 cm –1 , which is normally free of any protein bands.
3.3. Data Processing Techniques
3.3.1. IR Difference Spectroscopy
Difference spectroscopy involves the subtraction of a protein IR spectrum
in state A from that of the protein in state B. The resultant difference IR spectrum
only reveals features that are associated with those groups involved in a
conformational change. Figure 3 shows the infrared spectra of apo-calmodulin
(trace A) and of calmodulin saturated with Ca2+ (trace B). The two spectra are
dominated by a strong band centered at 1643/1644 cm –1 , which is a result of
the amide I’ mode of calmodulin. The amide II’ band is located at around
1455 cm –1 . Spectral features at 1550–1590 cm –1 arises from the antisymmetric
COO- stretching vibrations of the carboxylate moiety of the amino acid
side-chain groups of glutamate and aspartate; the correponding symmetric
bands are located at 1390–1430 cm –1 . The carboxylate modes are established
markers of metal-ion binding (6,7; see also Chapter 13, Volume 1). Calcium
binding results in an upshift of the symmetric stretching band and a downshift
of the antisymmetric band. In calmodulin, 14 out of the 38 COO- groups are
found in the Ca2+ -binding sites, and the features at 1430–1390 and 1550–1590
cm –1 in the infrared difference spectrum (see Fig. 3C) are highly characteristic
of the spectral changes associated with calcium binding to the carboxylate
ligands in calmodulin. Positive and negative features in the amide I’ region
suggest that only slight changes in secondary structure take place when Ca2+ binds to calmodulin.
3.3.2. Derivation Methods and Fourier Deconvolution
A major problem in the use of the amide I mode to secondary structure analysis
is that the infrared bands arising from each type of polypeptide conformation
are inherently broad and lie close together. This leads to only weakly
resolved broad features. Two mathematical procedures are very useful to help
identify overlapping components within the composite amide I band contour.
The first involves calculation of the nth derivative of the spectrum. Often, the
second derivative is calculated, which gives a negative peak for every band or