We previously performed DSC on the PDZ domain of the
tyrosine phosphatase PTPN3 (PTPN3-PDZ) [34] free or in complex with various PBMs known to interact with the PTPN3-PDZ. A
single endothermic peak was observed in the DSC thermograms of
PTPN3-PDZ free or complexed. The free domain showed a T m of
41
C, while Tm values between 45
C and 52
C were observed for
PTPN3-PDZ complexed to different PBMs. Thus, an increase of
4–11
C in the Tm was observed depending on the sequence of the
ligands, showing that the PBM binding onto PTPN3-PDZ stabilizes the domain in all complexes (Fig. 5a). A gain in thermal
stability is also observed for other PDZ domains such as MAST2PDZ, whose Tm increases from 47
C to 55–62
C upon PBM
binding (Fig. 5b). The ligand binding increases the Tm of the PDZ
domain, either by stabilizing the native conformation or by the
destabilization of the unfolded state.
3.4.4 NMR
Compared to other spectroscopic techniques, NMR sensitivity level
is low. To record spectra with a sufficient signal to noise (S/N) ratio
within rational acquisition time the sample needs to be quite concentrated. Currently, the common samples correspond to a volume
of 500 μl or 200 μl (5 mm or 3 mm tubes) containing 50–1000 μM
of a medium-sized protein (10–20 kDa). The sample buffer also
contains 1–5% (v/v) D 2 O to record a reference signal (lock signal)
to compensate for small changes/drifts of the magnetic field. The
NMR tube is cleaned with a soft tissue from the outside before it is
inserted into the instrument to remove anything spilled on the tube
or grease from the fingertips. When the sample is inserted into the
spectrometer and the temperature is equilibrated, the lock signal is
defined. The inhomogeneities in the magnetic field are then corrected (shim), and the impedance of the probes for each nucleus is
precisely adjusted. This is called “tuning the probe”. Finally, the
length of the 90
proton pulse is determined. The setup of the
experiment can be automated and can be achieved within
15–20 min. The quality of an NMR spectrum depends on the
S/N ratio and the resolution that are first related to the strength
of the static magnetic field. The S/N ratio also depends on the
concentration of the sample and the molecular mass and intrinsic
dynamics of the protein, which affect the linewidth of peaks in the
spectrum. The S/N ratio correlates with the number of FIDs
accumulated and is enhanced by using a cryoprobe. The resolution
of an NMR spectrum depends on the linewidth and the separation
of the resonances, which is directly correlated with the magnetic
field strength. Furthermore, the separation of the signals is determined by the number of data points collected in the respective
dimension.
A one-dimensional (1D)
1
H NMR spectrum of a protein
requires a less number of molecules and is fast (less than 1 min)
to perform and can unambiguously indicate if a protein is folded or
PDZ Sample Quality Assessment
117
tyrosine phosphatase PTPN3 (PTPN3-PDZ) [34] free or in complex with various PBMs known to interact with the PTPN3-PDZ. A
single endothermic peak was observed in the DSC thermograms of
PTPN3-PDZ free or complexed. The free domain showed a T m of
41
C, while Tm values between 45
C and 52
C were observed for
PTPN3-PDZ complexed to different PBMs. Thus, an increase of
4–11
C in the Tm was observed depending on the sequence of the
ligands, showing that the PBM binding onto PTPN3-PDZ stabilizes the domain in all complexes (Fig. 5a). A gain in thermal
stability is also observed for other PDZ domains such as MAST2PDZ, whose Tm increases from 47
C to 55–62
C upon PBM
binding (Fig. 5b). The ligand binding increases the Tm of the PDZ
domain, either by stabilizing the native conformation or by the
destabilization of the unfolded state.
3.4.4 NMR
Compared to other spectroscopic techniques, NMR sensitivity level
is low. To record spectra with a sufficient signal to noise (S/N) ratio
within rational acquisition time the sample needs to be quite concentrated. Currently, the common samples correspond to a volume
of 500 μl or 200 μl (5 mm or 3 mm tubes) containing 50–1000 μM
of a medium-sized protein (10–20 kDa). The sample buffer also
contains 1–5% (v/v) D 2 O to record a reference signal (lock signal)
to compensate for small changes/drifts of the magnetic field. The
NMR tube is cleaned with a soft tissue from the outside before it is
inserted into the instrument to remove anything spilled on the tube
or grease from the fingertips. When the sample is inserted into the
spectrometer and the temperature is equilibrated, the lock signal is
defined. The inhomogeneities in the magnetic field are then corrected (shim), and the impedance of the probes for each nucleus is
precisely adjusted. This is called “tuning the probe”. Finally, the
length of the 90
proton pulse is determined. The setup of the
experiment can be automated and can be achieved within
15–20 min. The quality of an NMR spectrum depends on the
S/N ratio and the resolution that are first related to the strength
of the static magnetic field. The S/N ratio also depends on the
concentration of the sample and the molecular mass and intrinsic
dynamics of the protein, which affect the linewidth of peaks in the
spectrum. The S/N ratio correlates with the number of FIDs
accumulated and is enhanced by using a cryoprobe. The resolution
of an NMR spectrum depends on the linewidth and the separation
of the resonances, which is directly correlated with the magnetic
field strength. Furthermore, the separation of the signals is determined by the number of data points collected in the respective
dimension.
A one-dimensional (1D)
1
H NMR spectrum of a protein
requires a less number of molecules and is fast (less than 1 min)
to perform and can unambiguously indicate if a protein is folded or
PDZ Sample Quality Assessment
117