Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

62 Fabian and Vogel
perature, ionic strength, pH) because slight variations will cause changes in the
spectral features of the H 2O or D 2O bands, thereby preventing an ideal subtraction
of the buffer contributions. For example, for measurements in water,
the temperatures of the sample in H 2O-solution and the buffer should coincide
within 0.1°C in order to avoid artifacts caused by temperature differences. The
subtraction of water from a protein spectrum requires a reference water band
that does not overlap with those of the sample. The weak combination band of
H 2O around 2126 cm –1 may serve as a good approximation to interactively
subtract the water features. The final water subtraction should be performed
using a different spectral region with stronger H 2O absorption, such as the one
in the vicinity of approx 3645 cm –1 (4).
3.2.1. Hydrogen–Deuterium Exchange, a Specific Feature of Protein
Studies in D 2O
The hydrogen–deuterium exchange of amide protons can be monitored by
the disappearence of the band characteristic of N–H bending near 1545 cm –1
(amide II) and the appearence of N–D absorption near 1455 cm –1 (amide II’).
The shift of the amide I band (predominantly C = O stretching vibration mode
of the amide group) upon deuteration of the backbone hydrogens (labeled
amide I’ by convention) is only small (5–10 cm –1 ). However, individual spectral
components of the amide I band of a protein often reveal different
exchange kinetics. This greatly assists the assignment of band components
arising from different secondary structural classes. Despite this positive
aspect, it can also complicate the interpretation of the amide I’ region, if a
protein cannot completely be exchanged. Figure 2A (solid line) shows the
infrared spectrum of the apo-form of calmodulin after complete H–D
exchange. The solvent spectrum (Fig. 2A, dotted line) was measured in an
optimally matched second cell of slightly reduced path length, which takes
into account the slightly lower D 2O concentration in the protein sample measured.
Figure 2B (solid line) shows the corresponding buffer-subtracted spectrum
of apo-calmodulin, together with a spectrum of the apo-form measured
15 min after dissolving the protein in D 2O (dotted line). Residual intensity at
3300 cm –1 (amide A; N–H stretching of the peptide groups) indicates that a
number of amide protons are not exchanged after short exposure of apocalmodulin
to D 2O. The amide A is the best indicator for nonexchanged N–H
groups because of the lack of other protein absorptions in the range 3200–
3400 cm –1 . The same information cannot easily be deduced from the residual
intensity in the amide II region, because infrared bands arising from amino
acid side-chain groups overlap with the remaining amide II band. For example,
in the case of apo-calmodulin, the carboxyl groups of aspartate and glutamate
residues absorb around 1575 cm –1 .