concentration (and thus reduce aggregation during denaturation)
and to ensure even heating. However, it is also possible to record
unfolding experiments in open (unstoppered) 1-mm cuvettes, e.g.,
when buffer absorption necessitates their use.
A temperature-controlled instrument will typically have a range
of 5–10
C through to 90–95
C. A denaturation experiment
should run from near the low- to near the high-temperature limit
of the instrument and subsequently cool back down to the starting
temperature, recording a full spectrum at the start and end. If the
CD spectrum following cooling is identical with the initial spectrum, this will indicate reversibility. If the unfolding reaction is
reversible, thermodynamic parameters may be determined
[19]. The wavelength is chosen where the difference between the
folded and unfolded protein (random coil) is large and of low noise
(222 nm for α-helical proteins and ~216 nm for beta-sheet-dominated structures).
For reasons of improving signal-to-noise (and thus enabling
low concentrations to be used), the CD signal is typically recorded
at a single wavelength during the unfolding process (although if the
instrument is sufficiently fast, recording full spectra has advantages
in analysis) [19].
A simple protocol for a thermal denaturation experiment is
given below:
Steps 1–6 as in section “Measurement of a CD Spectrum”
(above) to ensure that the cuvette is free from contamination.
7. Set the low- and high-temperature limits (e.g., 10 and 90
C)
and cool instrument to low-temperature limit.
8. Prepare the sample solution, centrifuge the sample in a benchtop centrifuge for 5–10 min at full speed, and degas it.
9. Fill a 1-cm cuvette with the sample solution. A buffer baseline is
not necessary as only differences between spectra will be
analyzed.
10. Place cuvette in holder, switch on stirrer (if applicable), and
allow sample time to cool to the low-temperature limit.
11. Record a full spectrum of the sample at the low-temperature
limit.
12. Select the wavelength for data recording where there is a large
signal and where the signal for a random coil (unfolded protein) will be small.
13. Heat the sample at 1
C/min. Record the CD signal at the
selected wavelength at closely spaced intervals. The temperature should be monitored via a temperature probe in the
cuvette.
14. After heating to 90
C, record a full spectrum of the sample.
34
Bertrand Raynal et al.
and to ensure even heating. However, it is also possible to record
unfolding experiments in open (unstoppered) 1-mm cuvettes, e.g.,
when buffer absorption necessitates their use.
A temperature-controlled instrument will typically have a range
of 5–10
C through to 90–95
C. A denaturation experiment
should run from near the low- to near the high-temperature limit
of the instrument and subsequently cool back down to the starting
temperature, recording a full spectrum at the start and end. If the
CD spectrum following cooling is identical with the initial spectrum, this will indicate reversibility. If the unfolding reaction is
reversible, thermodynamic parameters may be determined
[19]. The wavelength is chosen where the difference between the
folded and unfolded protein (random coil) is large and of low noise
(222 nm for α-helical proteins and ~216 nm for beta-sheet-dominated structures).
For reasons of improving signal-to-noise (and thus enabling
low concentrations to be used), the CD signal is typically recorded
at a single wavelength during the unfolding process (although if the
instrument is sufficiently fast, recording full spectra has advantages
in analysis) [19].
A simple protocol for a thermal denaturation experiment is
given below:
Steps 1–6 as in section “Measurement of a CD Spectrum”
(above) to ensure that the cuvette is free from contamination.
7. Set the low- and high-temperature limits (e.g., 10 and 90
C)
and cool instrument to low-temperature limit.
8. Prepare the sample solution, centrifuge the sample in a benchtop centrifuge for 5–10 min at full speed, and degas it.
9. Fill a 1-cm cuvette with the sample solution. A buffer baseline is
not necessary as only differences between spectra will be
analyzed.
10. Place cuvette in holder, switch on stirrer (if applicable), and
allow sample time to cool to the low-temperature limit.
11. Record a full spectrum of the sample at the low-temperature
limit.
12. Select the wavelength for data recording where there is a large
signal and where the signal for a random coil (unfolded protein) will be small.
13. Heat the sample at 1
C/min. Record the CD signal at the
selected wavelength at closely spaced intervals. The temperature should be monitored via a temperature probe in the
cuvette.
14. After heating to 90
C, record a full spectrum of the sample.
34
Bertrand Raynal et al.