In our example, since the interest is to understand the function
of different states of the S6-S7 loop of p53, we can use the p53
experimental structures of the DBD but also conformations of it
from out MD simulations with the loop in occluded or open states
(subjected to energy minimization before the PRISM analyses). We
identified a group of p53 interactors that are selectively bound to
the DBD with the S6-S7 loop in its open solvent accessible state, as
stated above and that have intriguing functional implication related
to transcription-independent function activities [14].
In summary, the protocol here suggested provides a rich portfolio of information that can be used for the design of experiments
to validate the modeling, and to disclose the mechanisms better. To
cite a few examples, the interactions predicted by PRISM could be
validated in vitro for example using chemical shift NMR perturbation experiments on the isolated domain of the protein or other
biophysical techniques suitable for studying protein complexes.
Moreover, the exchange by minor and major states that are modulated by DNA, PTMs, or mutations (such as the occluded and open
states of the S6-S7 loop) can be explored using NMR relaxation
dispersion measurements, also estimating from the exchange rate
the population of the states and directly comparing them to the
calculated ones. We could also exploit the protocol to design variants that can entrap one of the two states and experimentally test
our structural hypotheses.
4 Notes
1. In the case a CHARMM force field is used in Gromacs, it has to
be paid attention to the solvent model to select. The TIP3P
tailored for the CHARMM family [116] of the force field is not
necessarily the recommended option in some of the releases
and, it is the one labeled as “TIPS3P”. The simulations can
become slower, but if one is interested in solvent accessible
regions of the protein, the solvation is treated in a more suitable
way for the CHARMM family.
2. It is also always important to verify that there are very transient
or virtually none contacts between the periodic images, a suitable tool in this context is the g_mindist Gromacs tool.
3. If PCA is carried out using all atoms of the system, the covariance matrix of atomic fluctuations needs to be correctly massweighted.
4. A PyInteraph mass database compatible with the force field of
interest needs to be selected since different MD force fields
have different mass definitions.
Dynamics of p53
239
of different states of the S6-S7 loop of p53, we can use the p53
experimental structures of the DBD but also conformations of it
from out MD simulations with the loop in occluded or open states
(subjected to energy minimization before the PRISM analyses). We
identified a group of p53 interactors that are selectively bound to
the DBD with the S6-S7 loop in its open solvent accessible state, as
stated above and that have intriguing functional implication related
to transcription-independent function activities [14].
In summary, the protocol here suggested provides a rich portfolio of information that can be used for the design of experiments
to validate the modeling, and to disclose the mechanisms better. To
cite a few examples, the interactions predicted by PRISM could be
validated in vitro for example using chemical shift NMR perturbation experiments on the isolated domain of the protein or other
biophysical techniques suitable for studying protein complexes.
Moreover, the exchange by minor and major states that are modulated by DNA, PTMs, or mutations (such as the occluded and open
states of the S6-S7 loop) can be explored using NMR relaxation
dispersion measurements, also estimating from the exchange rate
the population of the states and directly comparing them to the
calculated ones. We could also exploit the protocol to design variants that can entrap one of the two states and experimentally test
our structural hypotheses.
4 Notes
1. In the case a CHARMM force field is used in Gromacs, it has to
be paid attention to the solvent model to select. The TIP3P
tailored for the CHARMM family [116] of the force field is not
necessarily the recommended option in some of the releases
and, it is the one labeled as “TIPS3P”. The simulations can
become slower, but if one is interested in solvent accessible
regions of the protein, the solvation is treated in a more suitable
way for the CHARMM family.
2. It is also always important to verify that there are very transient
or virtually none contacts between the periodic images, a suitable tool in this context is the g_mindist Gromacs tool.
3. If PCA is carried out using all atoms of the system, the covariance matrix of atomic fluctuations needs to be correctly massweighted.
4. A PyInteraph mass database compatible with the force field of
interest needs to be selected since different MD force fields
have different mass definitions.
Dynamics of p53
239