Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

60 Fabian and Vogel
The penetration depth of the infrared radiation in this arrangement is strictly
dependent on the wavelength and, therefore, the infrared spectrum measured
contains only information on a very thin layer of the sample that is in close
proximity to the surface of the crystal. This allows one to obtain a spectrum of
a protein in H 2O solution without much interference from infrared absorption
of the bulk water (1). Surface adsorption, however, may significantly change
the secondary structure of the protein molecules, which are in direct contact
with the crystal. Although the contribution of those moleclues to the total
absorbance measured might by small, one should proceed with caution in structural
studies of proteins by ATR spectroscopy (see Note 4).
2.2.5. Buffers and Denaturants
Fig. 1. Illustration of our custom-made IR cell.
Many commonly employed buffers, such as phosphate, cacodylate, Tris-
HCl, or HEPES, are acceptable. Buffers containing carboxylic acid groups,
such as acetate or carbonate buffers, are not ideal because their infrared
absorption bands overlap with those for the protein backbone.
Measurements of proteins in the presence of the calcium chelators EDTA or
EGTA are complicated by the fact that the carboxylic groups of these chelators
have strong infrared bands in the region 1570–1630 cm –1 . In addition, the spectral
characteristics of these bands are influenced by Ca 2+ -binding. As a consequence,
concentration and spectral features of the chelator in the sample cell
and the reference cell must be perfectly matched in order to avoid a misinterpretation
of the IR spectra between 1570–1630 cm –1 .
Obtaining infrared spectra of proteins in the presence of high concentrations
of the most commonly used denaturating agents, urea and guanidinium chloride
(GdmCl), is not simple. The strong infrared bands of urea around 1613 cm –1 or
GdmCl around 1600 cm –1 mask the much weaker protein amide I band. Isoto-