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Correlation spectroscopy and molecular dynamics simulations to study the structural features of proteins.

Varriale A, Marabotti A, Mei G, Staiano M, D'Auria S - PLoS ONE (2013)

Bottom Line: Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm(2)/s to 44±3 µm(2)/s when pH was changed from 7.0 to 4.0.An even more marked decrease of the MalE2 diffusion coefficient (31±3 µm(2)/s) was registered when pH was raised from 7.0 to 10.0.The obtained fluorescence correlation data, corroborated by circular dichroism, fluorescence emission and light-scattering experiments, are discussed together with the MD simulations results.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Sensing, IBP-CNR, Naples, Italy. a.varriale@ibp.cnr.it

ABSTRACT
In this work, we used a combination of fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulation methodologies to acquire structural information on pH-induced unfolding of the maltotriose-binding protein from Thermus thermophilus (MalE2). FCS has emerged as a powerful technique for characterizing the dynamics of molecules and it is, in fact, used to study molecular diffusion on timescale of microsecond and longer. Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm(2)/s to 44±3 µm(2)/s when pH was changed from 7.0 to 4.0. An even more marked decrease of the MalE2 diffusion coefficient (31±3 µm(2)/s) was registered when pH was raised from 7.0 to 10.0. According to the size of MalE2 (a monomeric protein with a molecular weight of 43 kDa) as well as of its globular native shape, the values of 44 µm(2)/s and 31 µm(2)/s could be ascribed to deformations of the protein structure, which enhances its propensity to form aggregates at extreme pH values. The obtained fluorescence correlation data, corroborated by circular dichroism, fluorescence emission and light-scattering experiments, are discussed together with the MD simulations results.

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Free energy landscape of MalE2.Projection of the trajectories of MalE2 at pH 4.0 (panel A), pH 7.0 (panel B) and pH 10.0 (panel C) in the essential plane defined by the two first eigenvectors calculated for the simulations. The probability is expressed as relative to the maximum frequency. The color scale (blue-red) defines the most probable conformation.
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pone-0064840-g007: Free energy landscape of MalE2.Projection of the trajectories of MalE2 at pH 4.0 (panel A), pH 7.0 (panel B) and pH 10.0 (panel C) in the essential plane defined by the two first eigenvectors calculated for the simulations. The probability is expressed as relative to the maximum frequency. The color scale (blue-red) defines the most probable conformation.

Mentions: Going down into the levels of protein structures, the analysis of the variation of secondary structures of the three systems made by DSSP shows that they are practically not influenced by the pH, since the content in secondary structures is nearly identical in the three conditions and is stable during the time of simulation. Also this result is in excellent agreement with far-UV CD data (data not shown). Therefore, it is confirmed also at molecular level that the perturbation produced by pH acts essentially on the overall tertiary fold of the protein. In order to gain insight into the protein flexibility and to identify large scale collective motions of atoms in MD simulations we performed a PCA on the three trajectories and identified the eigenvectors of the mass-weighted covariance matrix of the atomic positional fluctuations calculated on protein Cα atoms [38]. The results of PCA analysis suggests that probably no clear-cut collective motions are present during the simulation. The regions of the protein mainly affected by the motions described by the first two eigenvectors are the segments 25–50 and the segments 305–325 for simulations at pH 4.0, and the regions near residue 100 and 170 for simulations at pH 7.0 and pH 10.0. For each simulation at different pH, the results of PCA analysis were used to represent the free energy landscape (FEL) by projecting the MD trajectory on the essential plane defined by the first two eigenvectors (PC1 and PC2). Figure 7 shows the results of this calculation. At pH 7.0 there is a single hollow corresponding to the most probable conformation of this trajectory. On the contrary, at pH 4.0, at least three conformations are present, two of which with a deeper value of probability and with a narrow hollow. At pH 10.0 there are several minima, with two deeper hollows, less narrow than at pH 4.0. In conclusion, both analyses at pH 4.0 and pH 10.0 indicate the presence of multiple protein conformations probably due to the partial the unfolding of the MalE2 tertiary structure as a consequence of the induced perturbation of native ionic interactions.


Correlation spectroscopy and molecular dynamics simulations to study the structural features of proteins.

Varriale A, Marabotti A, Mei G, Staiano M, D'Auria S - PLoS ONE (2013)

Free energy landscape of MalE2.Projection of the trajectories of MalE2 at pH 4.0 (panel A), pH 7.0 (panel B) and pH 10.0 (panel C) in the essential plane defined by the two first eigenvectors calculated for the simulations. The probability is expressed as relative to the maximum frequency. The color scale (blue-red) defines the most probable conformation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672191&req=5

pone-0064840-g007: Free energy landscape of MalE2.Projection of the trajectories of MalE2 at pH 4.0 (panel A), pH 7.0 (panel B) and pH 10.0 (panel C) in the essential plane defined by the two first eigenvectors calculated for the simulations. The probability is expressed as relative to the maximum frequency. The color scale (blue-red) defines the most probable conformation.
Mentions: Going down into the levels of protein structures, the analysis of the variation of secondary structures of the three systems made by DSSP shows that they are practically not influenced by the pH, since the content in secondary structures is nearly identical in the three conditions and is stable during the time of simulation. Also this result is in excellent agreement with far-UV CD data (data not shown). Therefore, it is confirmed also at molecular level that the perturbation produced by pH acts essentially on the overall tertiary fold of the protein. In order to gain insight into the protein flexibility and to identify large scale collective motions of atoms in MD simulations we performed a PCA on the three trajectories and identified the eigenvectors of the mass-weighted covariance matrix of the atomic positional fluctuations calculated on protein Cα atoms [38]. The results of PCA analysis suggests that probably no clear-cut collective motions are present during the simulation. The regions of the protein mainly affected by the motions described by the first two eigenvectors are the segments 25–50 and the segments 305–325 for simulations at pH 4.0, and the regions near residue 100 and 170 for simulations at pH 7.0 and pH 10.0. For each simulation at different pH, the results of PCA analysis were used to represent the free energy landscape (FEL) by projecting the MD trajectory on the essential plane defined by the first two eigenvectors (PC1 and PC2). Figure 7 shows the results of this calculation. At pH 7.0 there is a single hollow corresponding to the most probable conformation of this trajectory. On the contrary, at pH 4.0, at least three conformations are present, two of which with a deeper value of probability and with a narrow hollow. At pH 10.0 there are several minima, with two deeper hollows, less narrow than at pH 4.0. In conclusion, both analyses at pH 4.0 and pH 10.0 indicate the presence of multiple protein conformations probably due to the partial the unfolding of the MalE2 tertiary structure as a consequence of the induced perturbation of native ionic interactions.

Bottom Line: Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm(2)/s to 44±3 µm(2)/s when pH was changed from 7.0 to 4.0.An even more marked decrease of the MalE2 diffusion coefficient (31±3 µm(2)/s) was registered when pH was raised from 7.0 to 10.0.The obtained fluorescence correlation data, corroborated by circular dichroism, fluorescence emission and light-scattering experiments, are discussed together with the MD simulations results.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Sensing, IBP-CNR, Naples, Italy. a.varriale@ibp.cnr.it

ABSTRACT
In this work, we used a combination of fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulation methodologies to acquire structural information on pH-induced unfolding of the maltotriose-binding protein from Thermus thermophilus (MalE2). FCS has emerged as a powerful technique for characterizing the dynamics of molecules and it is, in fact, used to study molecular diffusion on timescale of microsecond and longer. Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm(2)/s to 44±3 µm(2)/s when pH was changed from 7.0 to 4.0. An even more marked decrease of the MalE2 diffusion coefficient (31±3 µm(2)/s) was registered when pH was raised from 7.0 to 10.0. According to the size of MalE2 (a monomeric protein with a molecular weight of 43 kDa) as well as of its globular native shape, the values of 44 µm(2)/s and 31 µm(2)/s could be ascribed to deformations of the protein structure, which enhances its propensity to form aggregates at extreme pH values. The obtained fluorescence correlation data, corroborated by circular dichroism, fluorescence emission and light-scattering experiments, are discussed together with the MD simulations results.

Show MeSH