Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

FTIR Spectroscopy of Calcium-Binding Proteins 69
much as 15–20% of the total integrated intensity in this region. Sometimes the
infrared spectra of amino acids or simple peptides (10,16) are used for subtraction
of the side-chain contributions from the experimental protein spectrum.
The spectral parameters of the side-chain absorption bands in the model compounds,
however, provide only an approximation because the spectral features
of side-chain groups in a protein are influenced by the specific microenvironment
of the corresponding group.
3.4. Isotope-Edited FTIR Spectroscopy
The assignment of infrared bands to specific groups of a protein can be
accomplished by site-specific mutation or by isotopic labeling, and then by
comparing the spectra of the unmodified and the modified protein. Site-directed
mutagenesis can disturb the structure and function of the protein, thereby complicating
the assignments. Isotope substitution has the advantage of being
noninvasive, and facilitates band assignment by shifting bands that arise from
vibrational modes involving chemical groups, which contain the isotope.
Moreover, isotope labeling, such as site-specific 13C labeling of the polypeptide
backbone, allows FTIR spectroscopy to locate a particular secondary
structure within the polypeptide chain and helps analyzing conformational
changes that exclusively originate from the labeled site. The most valuable
way is site-directed isotope labeling, which is feasible without extra efforts by
chemical synthesis of peptides. The biosynthetically incorporation of a sitespecifically
labeled amino acid in a protein is much more difficult to achieve,
and have been reported for only very few proteins yet (17). What is easier to
achieve is uniform labeling of a specific type of amino acid residue in a protein.
As an example, we have incorporated 13C in the carbonyl position in the
polypeptide backbone of all methionine residues in calmodulin. As the
replacement of a 12C = O group with a 13C = O group decreases the amide I’
vibration by 35–45 cm –1 , a comparison of the spectra of the unlabeled and the
labeled protein allows the identification of the amide I’ bands that originate
from the labeled site. In the case of calmodulin, the amide I’ band at 1643 cm –1
undergoes a small drop in intensity, whereas a weak band assignable to the
13C-labeled carbonyls appears near 1600 cm –1 (compare the solid and dashed
line in Fig. 5A). Because the nine methionine residues in calmodulin are exclusively
located in helices, the observed isotopic shift provides direct evidence
for the assignment of the amide I’ band at 1643 cm –1 to α-helical structures
present in calmodulin.
Uniformly, 13C-labeling of a protein completely shifts the amide I’ band toward
lower wavenumbers (see Fig. 5B). By mixing a completely 13C = O-labeled
protein with an unlabeled peptide or protein, it then becomes feasible to observe
their respective amide I’ bands separately. This approach has been used to moni-