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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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Model of the 3D structure of MalE2 in the un-liganded form.The backbone of the protein is represented as a ribbon, and segments of secondary structures are shown as cylinders (helices) and arrows (strands).
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pone-0064840-g005: Model of the 3D structure of MalE2 in the un-liganded form.The backbone of the protein is represented as a ribbon, and segments of secondary structures are shown as cylinders (helices) and arrows (strands).

Mentions: The only available X-ray structure of MalE2 was obtained in complex with maltotriose [18] (PDB code: 2GH9). Unfortunately, this model is not very useful to discuss the observed pH-induced conformational changes in the maltotriose-free enzyme since the proteins belonging to the MalE2 family drastically change their tridimensional structure in the presence of the ligand [51]. We therefore decided to model in-silico the open and ligand-free form of MalE2 in order to simulate the behavior of the protein in the correct conformation. This strategy (as described in Material and Methods section) allowed us to obtain a reliable model using as reference the structure of MalE2 itself. We took advantage of the structure of the un-liganded form of the maltotriose-binding protein from T. maritima to model the connections between the two protein domains. The reciprocal orientation of the two protein domains in the open form (95.2% of the residues of the selected model) are in the most favored regions of the Ramachandran plot, with no residues in disallowed protein regions (the analysis performed on the template showed 93.7% and 0% of residues for most favored and disallowed regions, respectively). ProsaII z-score (−13.67) is in the range of scores typically found in proteins of similar sequence length [27] and it is similar to that of the template (−14.72). We also analyzed the energetic profile calculated by ProsaII on the whole protein structure, and our selected model displayed an optimal profile, with no positive peaks indicating errors in the structure (data not shown). These data demonstrate that our model is of high quality. Figure 5 shows the model of MalE2 in the protein un-liganded state.


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)

Model of the 3D structure of MalE2 in the un-liganded form.The backbone of the protein is represented as a ribbon, and segments of secondary structures are shown as cylinders (helices) and arrows (strands).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0064840-g005: Model of the 3D structure of MalE2 in the un-liganded form.The backbone of the protein is represented as a ribbon, and segments of secondary structures are shown as cylinders (helices) and arrows (strands).
Mentions: The only available X-ray structure of MalE2 was obtained in complex with maltotriose [18] (PDB code: 2GH9). Unfortunately, this model is not very useful to discuss the observed pH-induced conformational changes in the maltotriose-free enzyme since the proteins belonging to the MalE2 family drastically change their tridimensional structure in the presence of the ligand [51]. We therefore decided to model in-silico the open and ligand-free form of MalE2 in order to simulate the behavior of the protein in the correct conformation. This strategy (as described in Material and Methods section) allowed us to obtain a reliable model using as reference the structure of MalE2 itself. We took advantage of the structure of the un-liganded form of the maltotriose-binding protein from T. maritima to model the connections between the two protein domains. The reciprocal orientation of the two protein domains in the open form (95.2% of the residues of the selected model) are in the most favored regions of the Ramachandran plot, with no residues in disallowed protein regions (the analysis performed on the template showed 93.7% and 0% of residues for most favored and disallowed regions, respectively). ProsaII z-score (−13.67) is in the range of scores typically found in proteins of similar sequence length [27] and it is similar to that of the template (−14.72). We also analyzed the energetic profile calculated by ProsaII on the whole protein structure, and our selected model displayed an optimal profile, with no positive peaks indicating errors in the structure (data not shown). These data demonstrate that our model is of high quality. Figure 5 shows the model of MalE2 in the protein un-liganded state.

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