Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

FTIR Spectroscopy of Calcium-Binding Proteins 67
individual component bands can be resolved, whereby the relative integrated
intensities are maintained. Both band-narrowing techniques greatly amplify
features in the spectra originating from random noise and/or uncompensated
water vapor; they need to be used with great care to avoid artifacts (5,9). A
comparison of the deconvoluted spectra of the apo-form and the Ca 2+ form of
calmodulin given in Fig. 4A,B, respectively, reveals several band components
in the amide I’ region, which are hidden in the original spectrum of the corresponding
protein (see Fig. 3A,B). In addition, the visualization of the fine structure
of the side-chain absorptions of glutamate and aspartate is improved.
Moreover, weak bands at 1515/1516 and 1498/1499 cm –1 , which are caused by
amino acid side-chain absorptions (10) of tyrosine and phenylalanine, respectively,
can be identified in the deconvoluted spectra.
3.3.3. Curve Fitting of Band Contours
Curve fitting of amide I/I’ band profiles is often used to quantitatively analyze
underlying band components. In the curve-fitting approach, the number of
component bands estimated by Fourier self-deconvolution and derivative spectra,
plus their approximated width, height, and shape are used as input parameters
in an iterative least squares procedure that attempts to reproduce the
measured amide I/I’ band profile by varying these parameters. For practical
reasons, deconvoluted spectra should be subjected to curve fitting because
least-square algorithms are significantly more reliable for spectra with an
enhanced profile. When a reasonable fit is obtained, the fractional areas of the
fitted components are taken as directly proportional to the relative quantities of
the structure elements they represent. The percentages of different secondary
structure elements can then be estimated by adding the areas of all component
bands assigned to each of these structures and expressing the sum as a fraction
of the total amide I/I’ band area (11).
3.3.4. Problems Associated with the Curve-Fitting Procedure
The curve-fitting approach (like all curve-fitting applications) has some
inherent problems. An element of subjectivity is the assumption that the number
of band components estimated by self-deconvolution or derivation reflects the
real number of components. In cases where bands significantly overlap, even
the applied band-narrowing procedures certainly fail in separating the components
present. Another assumption in this method is that the molar absorptivities
of the bands associated with different secondary structural elements are
identical, which is at best a rough approximation (9). A very critical step is the
assignment of the component bands, which is based on theoretical calculations
and on emperical spectra-structure correlations experimentally established for
model polypeptides and proteins of known three-dimensional structure (9,11).