220
B. Czarnik-Matusewicz and Y.M. Jung
of a potential sequence of events in response to changes induced by a temperature
increase from 20 °C to 95 °C. using the procedure proposed by Noda [1, 6] for the
interpretation of the synchronous/asynchronous changes, three spectroscopic events
have been suggested for the RNase A system on this temperature scale; these events
involve structures that contribute to the following peaks: 1,637/1,692 cm
−1
(step 1),
1,656 cm
−1
(step 2), and 1,644 cm
−1
(step 3). Based on the well-defined relationship
between frequency and secondary structure [126], the spectral changes has been
“translated” into the following sequence: in the first step of the process, the partial
unfolding of the more strongly bonded β-sheet fragment occurred. In the next steps,
the α-helices and the weaker parts of the β-sheet unfold [38].
the thermally induced unfolding of RNase A has also been investigated under
reducing conditions maintained by the presence of 2-mercaptoethanol [83]. In these
studies fluorescence spectroscopy has also been employed, and 2dCoS was combined with principal component analysis (PCA) to interpret the infrared data. the
paper was published in a special volume of vibrational Spectroscopy dedicated to
Professor heinz Siesler, a pioneer in NIR spectroscopy of polymers and applications of 2dCoS for analysis.
Arrondo and coworkers have also frequently applied temperature as a perturbation mode. From temperature-perturbed infrared spectra of rat liver methionine
adenosyltransferase (mAt), the secondary structure and the events occurring during
the thermal unfolding of mAt were characterized [62]. First, based on the thermal activity profiles and one-dimensional analysis of the infrared and fluorescence
spectra, the unfolding and aggregation process of mAt was divided into three temperature stages. Next, analysis of the 2d correlation maps independently obtained
for each region indicated that the α-helix and β-turns change first. Aggregation,
which occurred at the expense of the α-helix, β-sheet, and the residual β-turns, a
major event in the second stage. Correlation maps corresponding to the highest
temperature range indicated no further major changes in protein conformation and
proved that denaturation is complete in the last stage.
In [65], urea and thermal treatments were combined to study the effect of urea
on the thermal denaturation of an integral membrane protein (sarcoplasmic reticulum Ca
2+
-AtPase) that pumps calcium out of the cytoplasm during the relaxation of
striated muscle. the action of the chaotropic agent urea on the thermal stability of
this protein was examined by temperature-perturbed 2dCoS for the control sample
(non-urea treated) and 3 m and 4 m solutions of urea. to check if the urea-induced
changes were reversible or non-reversible in character, the two urea systems were
washed twice with urea-free buffer and were heated; the obtained 2dCoS maps
were compared with those prior to washing and with the control sample. 2d-IRCoS spectroscopy revealed that in the 3 M system, the urea perturbed the α and β
structures and stopped the thermal transition in a reversible manner. however, in the
4 m urea system, the protein structure was changed in a non-reversible way.
temperature perturbation has also been used in studies of the influence of calcium on the stability of chicken annexin A5 (cA5), which can reversibly bind to
fatty acid phospholipid-rich membranes in the presence of calcium [59]. In temperature-dependent synchronous 2d correlation maps of cA5 and two mutants of
B. Czarnik-Matusewicz and Y.M. Jung
of a potential sequence of events in response to changes induced by a temperature
increase from 20 °C to 95 °C. using the procedure proposed by Noda [1, 6] for the
interpretation of the synchronous/asynchronous changes, three spectroscopic events
have been suggested for the RNase A system on this temperature scale; these events
involve structures that contribute to the following peaks: 1,637/1,692 cm
−1
(step 1),
1,656 cm
−1
(step 2), and 1,644 cm
−1
(step 3). Based on the well-defined relationship
between frequency and secondary structure [126], the spectral changes has been
“translated” into the following sequence: in the first step of the process, the partial
unfolding of the more strongly bonded β-sheet fragment occurred. In the next steps,
the α-helices and the weaker parts of the β-sheet unfold [38].
the thermally induced unfolding of RNase A has also been investigated under
reducing conditions maintained by the presence of 2-mercaptoethanol [83]. In these
studies fluorescence spectroscopy has also been employed, and 2dCoS was combined with principal component analysis (PCA) to interpret the infrared data. the
paper was published in a special volume of vibrational Spectroscopy dedicated to
Professor heinz Siesler, a pioneer in NIR spectroscopy of polymers and applications of 2dCoS for analysis.
Arrondo and coworkers have also frequently applied temperature as a perturbation mode. From temperature-perturbed infrared spectra of rat liver methionine
adenosyltransferase (mAt), the secondary structure and the events occurring during
the thermal unfolding of mAt were characterized [62]. First, based on the thermal activity profiles and one-dimensional analysis of the infrared and fluorescence
spectra, the unfolding and aggregation process of mAt was divided into three temperature stages. Next, analysis of the 2d correlation maps independently obtained
for each region indicated that the α-helix and β-turns change first. Aggregation,
which occurred at the expense of the α-helix, β-sheet, and the residual β-turns, a
major event in the second stage. Correlation maps corresponding to the highest
temperature range indicated no further major changes in protein conformation and
proved that denaturation is complete in the last stage.
In [65], urea and thermal treatments were combined to study the effect of urea
on the thermal denaturation of an integral membrane protein (sarcoplasmic reticulum Ca
2+
-AtPase) that pumps calcium out of the cytoplasm during the relaxation of
striated muscle. the action of the chaotropic agent urea on the thermal stability of
this protein was examined by temperature-perturbed 2dCoS for the control sample
(non-urea treated) and 3 m and 4 m solutions of urea. to check if the urea-induced
changes were reversible or non-reversible in character, the two urea systems were
washed twice with urea-free buffer and were heated; the obtained 2dCoS maps
were compared with those prior to washing and with the control sample. 2d-IRCoS spectroscopy revealed that in the 3 M system, the urea perturbed the α and β
structures and stopped the thermal transition in a reversible manner. however, in the
4 m urea system, the protein structure was changed in a non-reversible way.
temperature perturbation has also been used in studies of the influence of calcium on the stability of chicken annexin A5 (cA5), which can reversibly bind to
fatty acid phospholipid-rich membranes in the presence of calcium [59]. In temperature-dependent synchronous 2d correlation maps of cA5 and two mutants of