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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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Circular dichroism and fluorescence measurements.Panel A: Near-UV spectra of MalE2 at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue); Panel B: steady state fluorescence spectra of MalE2 at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue); inset: the ANS fluorescence spectra of MalE2 at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue); Panel C and D: lifetime distribution profiles obtained from dynamics fluorescence measurements at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue).
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pone-0064840-g004: Circular dichroism and fluorescence measurements.Panel A: Near-UV spectra of MalE2 at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue); Panel B: steady state fluorescence spectra of MalE2 at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue); inset: the ANS fluorescence spectra of MalE2 at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue); Panel C and D: lifetime distribution profiles obtained from dynamics fluorescence measurements at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue).

Mentions: Circular dichroism measurements in the far-UV yielded superimposable spectra of MalE2 at pH 7.0, pH 4.0 and 10.0, at 25°C (data not shown), indicating that alkaline and acidic pH do not have a measurable effect on the secondary structure content of MalE2. A similar result was almost expected, since also the secondary structure content of other α/βproteins resulted to be independent on pH variations [43], [44]. On the contrary, CD measurements in the aromatic region (250–300 nm) revealed that detectable spectral differences arise when the pH value is varied from 4.0 to 10.0 (Figure 4A). In particular, a decrease in the intensity of the typical absorption peaks of tryptophan residues (i.e. 280, 285 and 292 nm) is observed both at pH 4.0 and pH 10.0. This result indicates that the protein side chains are indeed more flexible at acid and alkaline pH values, suggesting a larger hydration of the MalE2 tertiary structure at pH 4.0 and pH 10.0 than at pH 7.0.


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)

Circular dichroism and fluorescence measurements.Panel A: Near-UV spectra of MalE2 at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue); Panel B: steady state fluorescence spectra of MalE2 at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue); inset: the ANS fluorescence spectra of MalE2 at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue); Panel C and D: lifetime distribution profiles obtained from dynamics fluorescence measurements at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672191&req=5

pone-0064840-g004: Circular dichroism and fluorescence measurements.Panel A: Near-UV spectra of MalE2 at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue); Panel B: steady state fluorescence spectra of MalE2 at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue); inset: the ANS fluorescence spectra of MalE2 at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue); Panel C and D: lifetime distribution profiles obtained from dynamics fluorescence measurements at pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue).
Mentions: Circular dichroism measurements in the far-UV yielded superimposable spectra of MalE2 at pH 7.0, pH 4.0 and 10.0, at 25°C (data not shown), indicating that alkaline and acidic pH do not have a measurable effect on the secondary structure content of MalE2. A similar result was almost expected, since also the secondary structure content of other α/βproteins resulted to be independent on pH variations [43], [44]. On the contrary, CD measurements in the aromatic region (250–300 nm) revealed that detectable spectral differences arise when the pH value is varied from 4.0 to 10.0 (Figure 4A). In particular, a decrease in the intensity of the typical absorption peaks of tryptophan residues (i.e. 280, 285 and 292 nm) is observed both at pH 4.0 and pH 10.0. This result indicates that the protein side chains are indeed more flexible at acid and alkaline pH values, suggesting a larger hydration of the MalE2 tertiary structure at pH 4.0 and pH 10.0 than at pH 7.0.

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