CD is particularly suited to the study of structural changes
induced by environmental variations (pH, temperature, detergents,
cosolvents, buffers, . . .). Among the advantages of CD compared to
other techniques, we can mention that one works in dilute solution,
with small amounts of product (a few tens micrograms), without
molecular size limit on the protein. The measurements are simple
and fast, allowing kinetic measurements at very short times. Moreover, the measurement is not destructive and it is possible to
recover the sample after the acquisition of the spectrum.
Figure 4 shows far-UV CD spectra of free MAST2-PDZ, free
PDZ-binding motif peptide and the complex MAST2-PDZ with a
PDZ-binding motif peptide in blue, orange, and gray, respectively.
The similarity of spectra of MAST2-PDZ free or complexed to a
PDZ ligand, in blue and gray respectively, is indicative of the
maintenance of the PDZ folding upon PDZ–ligand binding while
the peptide alone (in orange) is unfolded.
Through this technique, the thermal stability of a protein can
also be evaluated by looking at the CD shape of spectra recorded at
different temperatures; this operation can also be done continuously with a temperature spectrum at a fixed wavelength, at 222 nm
for example to follow the helical content of PDZ domains. Thermal
stability is now often evaluated by DSC or DSF (see the section
below).
Procedure for a standard CD acquisition;
1. Switch on the instrument and select the experimental
temperature.
2. Wait for temperature stabilization.
3. Clean the cell with water, dry with ethanol (with detergent,
e.g., 2%(v/v) Hellmanex, if needed).
4. Centrifuge the buffer using a benchtop centrifuge (15 min, at
full speed) to remove any aggregates.
5. Fill the cell with 40–50 μl of buffer. Avoid the formation of
bubbles.
6. Record ellipticity values every 0.5 nm, averaged over 2 s
between 195 and 260 nm. Three to five successive scans are
collected.
7. Clean again the cell with water, dry with ethanol.
8. Fill the cell with 40–50 μl of PDZ sample. Avoid the formation
of bubbles.
9. Collect the data with the same parameters as the ones used for
the buffer spectra.
10. The final spectrum of PDZ sample is obtained by averaging the
3–5 successive scans and subtracting the baseline spectrum of
the buffer.
PDZ Sample Quality Assessment
115
induced by environmental variations (pH, temperature, detergents,
cosolvents, buffers, . . .). Among the advantages of CD compared to
other techniques, we can mention that one works in dilute solution,
with small amounts of product (a few tens micrograms), without
molecular size limit on the protein. The measurements are simple
and fast, allowing kinetic measurements at very short times. Moreover, the measurement is not destructive and it is possible to
recover the sample after the acquisition of the spectrum.
Figure 4 shows far-UV CD spectra of free MAST2-PDZ, free
PDZ-binding motif peptide and the complex MAST2-PDZ with a
PDZ-binding motif peptide in blue, orange, and gray, respectively.
The similarity of spectra of MAST2-PDZ free or complexed to a
PDZ ligand, in blue and gray respectively, is indicative of the
maintenance of the PDZ folding upon PDZ–ligand binding while
the peptide alone (in orange) is unfolded.
Through this technique, the thermal stability of a protein can
also be evaluated by looking at the CD shape of spectra recorded at
different temperatures; this operation can also be done continuously with a temperature spectrum at a fixed wavelength, at 222 nm
for example to follow the helical content of PDZ domains. Thermal
stability is now often evaluated by DSC or DSF (see the section
below).
Procedure for a standard CD acquisition;
1. Switch on the instrument and select the experimental
temperature.
2. Wait for temperature stabilization.
3. Clean the cell with water, dry with ethanol (with detergent,
e.g., 2%(v/v) Hellmanex, if needed).
4. Centrifuge the buffer using a benchtop centrifuge (15 min, at
full speed) to remove any aggregates.
5. Fill the cell with 40–50 μl of buffer. Avoid the formation of
bubbles.
6. Record ellipticity values every 0.5 nm, averaged over 2 s
between 195 and 260 nm. Three to five successive scans are
collected.
7. Clean again the cell with water, dry with ethanol.
8. Fill the cell with 40–50 μl of PDZ sample. Avoid the formation
of bubbles.
9. Collect the data with the same parameters as the ones used for
the buffer spectra.
10. The final spectrum of PDZ sample is obtained by averaging the
3–5 successive scans and subtracting the baseline spectrum of
the buffer.
PDZ Sample Quality Assessment
115