13. At the end of the run, analyze all peaks of the chromatogram
for sample recovery and molecular mass determination according to the manufacturer’s manual. If the column type and the
buffer conditions are chosen correctly to avoid nonspecific
binding of the PDZ, you should recover nearly 100% of your
sample. The baseline for the detectors should be set up wisely,
that is, in a region where no proteins elute, such as the region
prior to the void volume of the column (first third of the
column volume). The baseline should remain constant if the
sample is in the same buffer as the elution buffer and if the
temperature is strictly controlled.
3.4 Conformational
Stability/Folding State
3.4.1 Circular
Dichroism (CD)
CD spectroscopy is a fast and easy method that is particularly
suitable for quickly judging the folding state of a protein; to compare the structure of proteins obtained from different sources; to
analyze the impact of point mutations on the structure of a protein;
to judge the stability of the structure facing environmental changes
(pH, salinity); to determine the impact on the structure in the
presence of a ligand.
One important point to improve the quality of the result is to
use well adapted buffer; it must not have an absorbance higher than
1 in the range of the spectrum (see Note 21). CD spectrometers
typically measure from longer to shorter wavelengths. The combination of accumulations and the time over which the instrument
averages each data point needs to be optimized to get the best
signal to noise (S/N) ratio. The parameters of a measurement
should be carefully chosen [20]. Usually, the CD signal, that is,
the ellipticity θ, the high voltage, and the absorbance are recorded.
In order to obtain reliable, interpretable, and high-quality CD data,
the absorbance of the sample (including buffer) should be between
1 and 2, that is, on most laboratory instruments the high voltage
should be kept below 600 V. A CD spectrum of the buffer is
recorded with the same set of parameters that will be applied in
the sample measurement to allow a baseline subtraction. A CD
spectrum recorded in the 180–260 nm range allows to evaluate
the secondary structure content and then the folding state of the
protein. The resulting spectrum has a characteristic shape and
magnitude determined by the secondary structure elements. The
fraction of each secondary structure element (α-helices, β-sheets,
turns, and disordered structures) is evaluated using different algorithms combining a set of reference spectra [43]. Dichroic signals in
the near UV (250–330 nm, absorption zone of aromatic amino
acids) provide information on the environment of these aromatic
amino acids and are a signature of tertiary and quaternary structure.
In these wavelengths, the CD signals of proteins are weak; cuvettes
with larger pathlengths and higher concentrations of PDZ sample
are required.
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Ce ´ lia Caillet-Saguy et al.
for sample recovery and molecular mass determination according to the manufacturer’s manual. If the column type and the
buffer conditions are chosen correctly to avoid nonspecific
binding of the PDZ, you should recover nearly 100% of your
sample. The baseline for the detectors should be set up wisely,
that is, in a region where no proteins elute, such as the region
prior to the void volume of the column (first third of the
column volume). The baseline should remain constant if the
sample is in the same buffer as the elution buffer and if the
temperature is strictly controlled.
3.4 Conformational
Stability/Folding State
3.4.1 Circular
Dichroism (CD)
CD spectroscopy is a fast and easy method that is particularly
suitable for quickly judging the folding state of a protein; to compare the structure of proteins obtained from different sources; to
analyze the impact of point mutations on the structure of a protein;
to judge the stability of the structure facing environmental changes
(pH, salinity); to determine the impact on the structure in the
presence of a ligand.
One important point to improve the quality of the result is to
use well adapted buffer; it must not have an absorbance higher than
1 in the range of the spectrum (see Note 21). CD spectrometers
typically measure from longer to shorter wavelengths. The combination of accumulations and the time over which the instrument
averages each data point needs to be optimized to get the best
signal to noise (S/N) ratio. The parameters of a measurement
should be carefully chosen [20]. Usually, the CD signal, that is,
the ellipticity θ, the high voltage, and the absorbance are recorded.
In order to obtain reliable, interpretable, and high-quality CD data,
the absorbance of the sample (including buffer) should be between
1 and 2, that is, on most laboratory instruments the high voltage
should be kept below 600 V. A CD spectrum of the buffer is
recorded with the same set of parameters that will be applied in
the sample measurement to allow a baseline subtraction. A CD
spectrum recorded in the 180–260 nm range allows to evaluate
the secondary structure content and then the folding state of the
protein. The resulting spectrum has a characteristic shape and
magnitude determined by the secondary structure elements. The
fraction of each secondary structure element (α-helices, β-sheets,
turns, and disordered structures) is evaluated using different algorithms combining a set of reference spectra [43]. Dichroic signals in
the near UV (250–330 nm, absorption zone of aromatic amino
acids) provide information on the environment of these aromatic
amino acids and are a signature of tertiary and quaternary structure.
In these wavelengths, the CD signals of proteins are weak; cuvettes
with larger pathlengths and higher concentrations of PDZ sample
are required.
114
Ce ´ lia Caillet-Saguy et al.