Article pubs.acs.org/JPCB
Influence of Pressure and Crowding on the Sub-Nanosecond Dynamics of Globular Proteins M. Erlkamp,† J. Marion,‡,§ N. Martinez,‡,§ C. Czeslik,† J. Peters,‡,§ and R. Winter*,† †
Physical Chemistry I − Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Str. 6, 44227 Dortmund, Germany ‡ Université Grenoble Alpes, IBS, 71 avenue des Martyrs, CS 10090, 38044 Grenoble, France § Institut Laue-Langevin, 71 avenue des Martyrs, CS 20156, 38042 CEDEX 9 Grenoble, France ABSTRACT: The influence of high hydrostatic pressure on the internal sub-nanosecond dynamics of highly concentrated lysozyme in aqueous solutions was studied by elastic incoherent neutron scattering (EINS) up to pressures of 4 kbar. We have found, with increasing pressure, a reduction in the dynamics of H atoms of folded lysozyme, suggesting a loss in protein mobility that follows a change in the local energy landscape upon the increase in packing density. Moreover, the amplitude of the protein fluctuations depends drastically on the protein concentration, and protein structural and interaction parameters as well as the dynamical properties are affected by pressure in a nonlinear way. A significant reduction of the mean squared displacement of H atoms occurs already at rather low pressures of a few hundred bars for lysozyme in bulk water solution. This trend is lifted at ∼2 kbar, which is probably due to a solvent-mediated effect. Conversely, for high protein concentrations (e.g., 160 mg mL−1), that is, under strong self-crowding conditions, as they are also encountered in the biological cell, strong restriction of the dynamics of protein motions takes place, reducing the mean squared displacement of H atoms by 60% and rendering its pressure dependence almost negligible. These results are also important for understanding the pressure stability of highly concentrated protein solutions in organisms thriving under hydrostatic pressure conditions such as in the deep sea, where pressures up to the kbar level are reached.
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INTRODUCTION Owing to their intrinsically low thermodynamic stability against unfolding (generally with free energy changes ΔG° < 20−40 kJ mol−1), proteins are sensitive to small variations in their environmental conditions, such as temperature, pressure, hydration, pH, ionic strength, and the presence of cosolutes.1 Whereas the effect of temperature on proteins is rather well documented, that of pressure is comparatively less studied.2−10 A thorough understanding of the stability of proteins requires the study of both, temperature and pressure effects, however. Moreover, in the present view of proteins as dynamic objects, which encompasses internal motions to fulfill their specific functions, it became also clear that it is crucial to study the effect of pressure on protein dynamics as well.11,12 Such studies, both theoretical and experimental, are still scarce.13−18 What is known so far, is that high hydrostatic pressure (HHP) reduces atomic fluctuations of proteins and slows down their diffusive and relaxational motions in the kbar range.13,14 Here we discuss pressure conditions, where the proteins are still in their natively folded state. Most globular proteins unfold at pressures above about 4−6 kbar.2−10 From a thermodynamic point of view, the extensive conjugate variable related to pressure is the volume, and changing the pressure exerted on a system permits a fine-tuned © XXXX American Chemical Society
exploration of its energy landscape through small volume changes. Moreover, fluctuations in volume have been shown to inevitably influence the molecular length scale and lead to variations in both the topology and the dissipation characteristics.14 For amino acid residues of globular proteins, these fluctuations occur on the sub-nanosecond time scale. Therefore, pressure-induced variations in the protein energy landscape may be important for substate alterations and hence of functional relevance.14 Moreover, investigating pressure effects is also of relevance for understanding the behavior of biological matter under extreme environmental conditions, such as in the deep sea where pressures up to the kbar level (1 kbar = 100 MPa) and beyond are encountered.19,20 From a cell biology point of view, there is an increasing interest in studies of the behavior of proteins in highly crowded solutions to mimic intracellular environments.21−24 A distinctive property of the biological cell is that its chemical processes proceed in a medium, whose volume is occupied by proteins and other biopolymers to an extent of about 20−30%. Such Received: January 31, 2015 Revised: March 11, 2015
A
DOI: 10.1021/acs.jpcb.5b01017 J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B conditions are able to cause significant alterations of the conformational stability and biological activity of proteins. Knowledge of protein−protein interactions in condensed fluid phases is also crucial for understanding processes like protein crystallization, aggregation, and fibrillation.25−33 Owing to the importance of such crowding studies, we set out to explore the effect of self-crowding, that is, high protein concentrations, on the sub-nanosecond dynamics of the protein lysozyme upon compression up to pressures of 4 kbar. Using small-angle X-ray scattering (SAXS) in combination with liquid-state theoretical approaches, we have already studied the structural properties of lysozyme and its intermolecular interactions at high concentration and high hydrostatic pressure.25−28 From the SAXS intensities as a function of the momentum transfer Q, I(Q), the attractive part of the interaction potential, J, could be derived by fitting a modified DLVO (Derjaguin−Landau−Verwey−Overbeek) model in the mean spherical approximation (MSA) to the structure factor data.25 The appearance of a correlation peak, at momentum transfer Qcorr, which originates from repulsive protein−protein interaction of the highly positively charged (protein charge Z = 8) lysozyme molecules, is clearly visible around 0.7 nm−1. Remarkably, as depicted in the inset of Figure 1, a more or less shallow maximum of Qcorr(p) as a function of
In contrast, information about the dynamics under such selfcrowding conditions, in particular also at high pressure conditions, is still lacking. On short time scales (
Influence of Pressure and Crowding on the Sub-Nanosecond Dynamics of Globular Proteins M. Erlkamp,† J. Marion,‡,§ N. Martinez,‡,§ C. Czeslik,† J. Peters,‡,§ and R. Winter*,† †
Physical Chemistry I − Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Str. 6, 44227 Dortmund, Germany ‡ Université Grenoble Alpes, IBS, 71 avenue des Martyrs, CS 10090, 38044 Grenoble, France § Institut Laue-Langevin, 71 avenue des Martyrs, CS 20156, 38042 CEDEX 9 Grenoble, France ABSTRACT: The influence of high hydrostatic pressure on the internal sub-nanosecond dynamics of highly concentrated lysozyme in aqueous solutions was studied by elastic incoherent neutron scattering (EINS) up to pressures of 4 kbar. We have found, with increasing pressure, a reduction in the dynamics of H atoms of folded lysozyme, suggesting a loss in protein mobility that follows a change in the local energy landscape upon the increase in packing density. Moreover, the amplitude of the protein fluctuations depends drastically on the protein concentration, and protein structural and interaction parameters as well as the dynamical properties are affected by pressure in a nonlinear way. A significant reduction of the mean squared displacement of H atoms occurs already at rather low pressures of a few hundred bars for lysozyme in bulk water solution. This trend is lifted at ∼2 kbar, which is probably due to a solvent-mediated effect. Conversely, for high protein concentrations (e.g., 160 mg mL−1), that is, under strong self-crowding conditions, as they are also encountered in the biological cell, strong restriction of the dynamics of protein motions takes place, reducing the mean squared displacement of H atoms by 60% and rendering its pressure dependence almost negligible. These results are also important for understanding the pressure stability of highly concentrated protein solutions in organisms thriving under hydrostatic pressure conditions such as in the deep sea, where pressures up to the kbar level are reached.
■
INTRODUCTION Owing to their intrinsically low thermodynamic stability against unfolding (generally with free energy changes ΔG° < 20−40 kJ mol−1), proteins are sensitive to small variations in their environmental conditions, such as temperature, pressure, hydration, pH, ionic strength, and the presence of cosolutes.1 Whereas the effect of temperature on proteins is rather well documented, that of pressure is comparatively less studied.2−10 A thorough understanding of the stability of proteins requires the study of both, temperature and pressure effects, however. Moreover, in the present view of proteins as dynamic objects, which encompasses internal motions to fulfill their specific functions, it became also clear that it is crucial to study the effect of pressure on protein dynamics as well.11,12 Such studies, both theoretical and experimental, are still scarce.13−18 What is known so far, is that high hydrostatic pressure (HHP) reduces atomic fluctuations of proteins and slows down their diffusive and relaxational motions in the kbar range.13,14 Here we discuss pressure conditions, where the proteins are still in their natively folded state. Most globular proteins unfold at pressures above about 4−6 kbar.2−10 From a thermodynamic point of view, the extensive conjugate variable related to pressure is the volume, and changing the pressure exerted on a system permits a fine-tuned © XXXX American Chemical Society
exploration of its energy landscape through small volume changes. Moreover, fluctuations in volume have been shown to inevitably influence the molecular length scale and lead to variations in both the topology and the dissipation characteristics.14 For amino acid residues of globular proteins, these fluctuations occur on the sub-nanosecond time scale. Therefore, pressure-induced variations in the protein energy landscape may be important for substate alterations and hence of functional relevance.14 Moreover, investigating pressure effects is also of relevance for understanding the behavior of biological matter under extreme environmental conditions, such as in the deep sea where pressures up to the kbar level (1 kbar = 100 MPa) and beyond are encountered.19,20 From a cell biology point of view, there is an increasing interest in studies of the behavior of proteins in highly crowded solutions to mimic intracellular environments.21−24 A distinctive property of the biological cell is that its chemical processes proceed in a medium, whose volume is occupied by proteins and other biopolymers to an extent of about 20−30%. Such Received: January 31, 2015 Revised: March 11, 2015
A
DOI: 10.1021/acs.jpcb.5b01017 J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B conditions are able to cause significant alterations of the conformational stability and biological activity of proteins. Knowledge of protein−protein interactions in condensed fluid phases is also crucial for understanding processes like protein crystallization, aggregation, and fibrillation.25−33 Owing to the importance of such crowding studies, we set out to explore the effect of self-crowding, that is, high protein concentrations, on the sub-nanosecond dynamics of the protein lysozyme upon compression up to pressures of 4 kbar. Using small-angle X-ray scattering (SAXS) in combination with liquid-state theoretical approaches, we have already studied the structural properties of lysozyme and its intermolecular interactions at high concentration and high hydrostatic pressure.25−28 From the SAXS intensities as a function of the momentum transfer Q, I(Q), the attractive part of the interaction potential, J, could be derived by fitting a modified DLVO (Derjaguin−Landau−Verwey−Overbeek) model in the mean spherical approximation (MSA) to the structure factor data.25 The appearance of a correlation peak, at momentum transfer Qcorr, which originates from repulsive protein−protein interaction of the highly positively charged (protein charge Z = 8) lysozyme molecules, is clearly visible around 0.7 nm−1. Remarkably, as depicted in the inset of Figure 1, a more or less shallow maximum of Qcorr(p) as a function of
In contrast, information about the dynamics under such selfcrowding conditions, in particular also at high pressure conditions, is still lacking. On short time scales (
