Classical molecular dynamics (MD) simulations, within the AMBER program package that runs entirely on a CUDA-enabled NVIDIA graphic processing unit (GPU), were employed to study the dynamics of the methane-thiosulfonate spin labelled (MTSL)  Aurora-A kinase activation loop in a very short time and with good quality of the sampling. The MD simulation provided a wealth of information on the interactions between MTSL and protein residues, and on the different motional contributions to the overall dynamics of the MTSL that were validated using a multifrequency electron paramagnetic resonance (EPR) approach. The latter relayed on the frequency dependence of the resolution of the fast and slow motions of the spin probe and was used to distinguish the fast internal motion of the spin label from the slow protein tumbling. Data obtained from MD were in good agreement with those obtained from quantum mechanical (QM) methods, but more interactions within the dynamics of the system were revealed than from QM. A strong correlation between the tumbling of the protein and the transitions of the X4 dihedral angle of the MTSL was observed with a consequent effect on the distribution of the nitroxide (NO) group in space and time . The theoretical  EPR spectra were calculated using selected configurations of MTSL probing different micro-environments of the protein characterized by different polarity. The comparison between the theoretical and experimental 9 GHz and 94 GHz EPR spectra revealed that some fits were in good agreement with the experimental EPR spectra, indicating a predominance of some conformational states of the full spin-labelled system. This work is a starting point for deeper experimental and theoretical studies of the diffusion properties of the Aurora-A kinase protein related to its overall tumbling and biological activity .
Classical molecular dynamics (MD) simulations, within the AMBER program package that runs entirely on a CUDA-enabled NVIDIA graphic processing unit (GPU), were employed to study with low computational cost and good quality of the sampling the dynamics of the methane-thiosulfonate spin label (MTSL) attached to the activation loop of the Aurora-A kinase. MD provided a wealth of information about the timescale of the different motional contributions to the overall dynamics of the spin label. These data were validated by multi-frequency continuous-wave electron paramagnetic resonance (EPR) measurements, that relying on the frequency dependence of the fast and slow motions of the spin probe were used to distinguish the fast internal motion of the spin label from slow protein tumbling. It was found that the activation loop oscillated between two conformational states separated by 7 Angstrom and the average structures obtained from the MD trajectories showed the MTSL exposed to the solvent and probing the C-lobe of the protein . The theoretical 9 and 94 GHz EPR spectra were calculated using configurations representing the interactions between MTSL and water and the tyrosine residue 208 in the C-lobe; and the comparison with experimental EPR spectra revealed that fits successfully reproduced the experimental spectra in agreement with the MD results .
R
the
with low computational cost and good quality of the sampling the
R
labelled
label
A
attached to the activation loop of the
R
kinase activation loop in a very short time and with good quality of the sampling. The MD simulation
kinase. MD
R
on the interactions between MTSL and protein residues, and on
about the timescale of
R
MTSL that were validated using a multifrequency
spin label. These data were validated by multi-frequency continuous-wave
R
approach. The latter relayed
measurements, that relying
D
resolution of the
R
and was
were
D
the
R
Data obtained from MD were in good agreement with those obtained from quantum mechanical (QM) methods, but more interactions within the dynamics of the system were revealed than from QM. A strong correlation between the tumbling of the protein and the transitions of the X4 dihedral angle of the MTSL was observed with a consequent effect on the distribution of the nitroxide (NO) group in space and time
It was found that the activation loop oscillated between two conformational states separated by 7 Angstrom and the average structures obtained from the MD trajectories showed the MTSL exposed to the solvent and probing the C-lobe of the protein
A
9 and 94 GHz
R
selected configurations of MTSL probing different micro-environments of the protein characterized by different polarity. The comparison between the theoretical and experimental 9 GHz and 94 GHz
configurations representing the interactions between MTSL and water and the tyrosine residue 208 in the C-lobe; and the comparison with experimental
R
some fits were in good
fits successfully reproduced the experimental spectra in
R
experimental EPR spectra, indicating a predominance of some conformational states of the full spin-labelled system. This work is a starting point for deeper experimental and theoretical studies of the diffusion properties of the Aurora-A kinase protein related to its overall tumbling and biological activity
MD results