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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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Dynamic light scattering measurements.Particle size distribution as a function of pH of MalE2 at pH 2.0 (black), pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue). The measurements were performed at 25°C. The final concentration of protein was 0.1 mg/ml.
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pone-0064840-g003: Dynamic light scattering measurements.Particle size distribution as a function of pH of MalE2 at pH 2.0 (black), pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue). The measurements were performed at 25°C. The final concentration of protein was 0.1 mg/ml.

Mentions: Light scattering measurements (Figure 3) also indicate that at pH 7.0 the distribution of the MalE2 molecules radius in solution is centered around 28 Å, confirming that the Stokes-Einstein model is a good approximation in the case of the native protein. On the contrary, using the diffusion coefficients obtained both in acid (pH 4.0) and basic (pH 10.0) conditions yielded (through equation 3) unreliable RSph values (57 and 81 Å, respectively), much larger than those observed in the light scattering measurements (Figure 3). Such a discrepancy suggests that: i) in these conditions the system is probably more heterogeneous than at pH 7.0, and ii) a different model must be applied to explain the decrease of the D1 value. The first hypothesis is supported by both light scattering (Figure 3) and FCS (Figure 2) measurements, which indicate a progressive increase in the protein aggregation propensity, as the pH is changed from pH 7.0 to pH 2.0 or pH 10.0.


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)

Dynamic light scattering measurements.Particle size distribution as a function of pH of MalE2 at pH 2.0 (black), pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue). The measurements were performed at 25°C. The final concentration of protein was 0.1 mg/ml.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0064840-g003: Dynamic light scattering measurements.Particle size distribution as a function of pH of MalE2 at pH 2.0 (black), pH 4.0 (red), pH 7.0 (green) and pH 10.0 (blue). The measurements were performed at 25°C. The final concentration of protein was 0.1 mg/ml.
Mentions: Light scattering measurements (Figure 3) also indicate that at pH 7.0 the distribution of the MalE2 molecules radius in solution is centered around 28 Å, confirming that the Stokes-Einstein model is a good approximation in the case of the native protein. On the contrary, using the diffusion coefficients obtained both in acid (pH 4.0) and basic (pH 10.0) conditions yielded (through equation 3) unreliable RSph values (57 and 81 Å, respectively), much larger than those observed in the light scattering measurements (Figure 3). Such a discrepancy suggests that: i) in these conditions the system is probably more heterogeneous than at pH 7.0, and ii) a different model must be applied to explain the decrease of the D1 value. The first hypothesis is supported by both light scattering (Figure 3) and FCS (Figure 2) measurements, which indicate a progressive increase in the protein aggregation propensity, as the pH is changed from pH 7.0 to pH 2.0 or pH 10.0.

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