Pressure-dependent electronic structure calculations using integral equation-based solvation models.

Recent methodological progress in quantum-chemical calculations using the "embedded cluster reference interaction site model" (EC-RISM) integral equation theory is reviewed in the context of applying it as a solvation model for calculating pressure-dependent thermodynamic and spectroscopic properties of molecules immersed in water. The methodology is based on self-consistent calculations of electronic and solvation structure around dissolved molecules where pressure enters the equations via an appropriately chosen solvent response function and the pure solvent density. Besides specification of a dispersion-repulsion force field for solute-solvent interactions, the EC-RISM approach derives the electrostatic interaction contributions directly from the wave function. We further develop and apply the method to a variety of benchmark cases for which computational or experimental reference data are either available in the literature or are generated specifically for this purpose in this work. Starting with an enhancement to predict hydration free energies at non-ambient pressures, which is the basis for pressure-dependent molecular population estimation, we demonstrate the performance on the calculation of the autoionization constant of water. Spectroscopic problems are addressed by studying the biologically relevant small osmolyte TMAO (trimethylamine N-oxide). Pressure-dependent NMR shifts are predicted and compared to experiments taking into account proper computational referencing methods that extend earlier work. The experimentally observed IR blue-shifts of certain vibrational bands of TMAO as well as of the cyanide anion are reproduced by novel methodology that allows for weighing equilibrium and non-equilibrium solvent relaxation effects. Taken together, the model systems investigated allow for an assessment of the reliability of the EC-RISM approach for studying pressure-dependent biophysical processes.

Osmolytes modify protein dynamics and function of tetrameric lactate dehydrogenase upon pressurization.

We present a study of the combined effects of natural cosolvents (TMAO, glycine, urea) and pressure on the activity of the tetrameric enzyme lactate dehydrogenase (LDH). To this end, high-pressure stopped-flow methodology in concert with fast UV/Vis spectroscopic detection of product formation was applied. To reveal possible pressure effects on the stability and dynamics of the enzyme, FTIR spectroscopic and neutron scattering measurements were carried out. In neat buffer solution, the catalytic turnover number of the enzyme, kcat, increases up to 1000 bar, the pressure range where dissociation of the tetrameric species to dimers sets in. Accordingly, we obtain a negative activation volume, ΔV# = -45.3 mL mol-1. Further, the enzyme substrate complex has a larger volume compared to the enzyme and substrate in the unbound state. The neutron scattering data show that changes in the fast internal dynamics of the enzyme are not responsible for the increase of kcat upon compression. Whereas the magnitude of kcat is similar in the presence of the osmolytes, the pressure of deactivation is modulated by the addition of cosolvents. TMAO and glycine increase the pressure of deactivation, and in accordance with the observed stabilizing effect both cosolvents exhibit against denaturation and/or dissociation of proteins. While urea does not markedly affect the magnitude of the Michaelis constant, KM, both 1 M TMAO and 1 M glycine exhibit smaller KM values of about 0.07 mM and 0.05 mM below about 1 kbar. Such positive effect on the substrate affinity could be rationalized by the effect the two cosolutes impose on the thermodynamic activities of the reactants, which reflect changes in water-mediated intermolecular interactions. Our data show that the intracellular milieu, i.e., the solution conditions that have evolved, may be sufficient to maintain enzymatic activity under extreme environmental conditions, including the whole pressure range encountered on Earth.

Cosolvent and Crowding Effects on the Temperature- and Pressure-Dependent Dissociation Process of the α/ß-Tubulin Heterodimer.

Tubulin is one of the main components of the cytoskeleton of eukaryotic cells. The formation of microtubules depends strongly on environmental and solution conditions, and has been found to be among the most pressure sensitive processes in vivo. We explored the effects of different types of cosolvents, such as trimethylamine-N-oxide (TMAO), sucrose and urea, and crowding agents to mimic cell-like conditions, on the temperature and pressure stability of the building block of microtubules, i. e. the α/ß-tubulin heterodimer. To this end, fluorescence and FTIR spectroscopy, differential scanning and pressure perturbation calorimetry as well as fluorescence anisotropy and correlation spectroscopies were applied. The pressure and temperature of dissociation of α/ß-tubulin as well as the underlying thermodynamic parameters upon dissociation, such as volume and enthalpy changes, have been determined for the different solution conditions. The temperature and pressure of dissociation of the α/ß-tubulin heterodimer and hence its stability increases dramatically in the presence of TMAO and the nanocrowder sucrose. We show that by adjusting the levels of compatible cosolutes and crowders, cells are able to withstand deteriorating effects of pressure even up to the kbar-range.

Exploring the influence of natural cosolvents on the free energy and conformational landscape of filamentous actin and microtubules.

Actin and tubulin, the main components of the cytoskeleton, are responsible for many different cellular functions and can be found in nearly all eukaryotic cells. The formation of filamentous actin (F-actin) as well as microtubules depends strongly on environmental and solution conditions. The self-assembly of both, actin and tubulin, has been found to be among the most pressure sensitive process in vivo. Here, we explored the effects of various types of natural cosolvents, such as urea and the osmolyte trimethylamine-N-oxide (TMAO), on the temperature- and pressure-dependent stability of their polymeric states, F-actin and microtubules. Accumulation of TMAO by deep-sea animals is proposed to protect against destabilizing effects of pressure. The pressure and temperature of unfolding as well as associated enthalpy and volume changes have been determined using Fourier-transform infrared spectroscopy, covering a wide range of pressures and temperatures, ranging from 1 bar to 11 kbar and from 20 to 90 °C, respectively. Complementary thermodynamic measurements have been carried out using differential scanning and pressure perturbation calorimetry. The results obtained helped us explore the effect of the cellular milieu on the limitations of the pressure stability of cytoskeletal assemblies. Conversely to urea, the pressure stability of both polymers increases dramatically in the presence of TMAO, counteracting detrimental effects of both, urea and pressure.

Antagonistic effects of natural osmolyte mixtures and hydrostatic pressure on the conformational dynamics of a DNA hairpin probed at the single-molecule level.

Organisms are thriving in the deep sea at pressures of up to the 1 kbar level. To withstand such harsh conditions, they accumulate particular osmolyte mixtures to counteract the pressure stress imposed. We explored the combined effects of pressure and osmolyte mixtures known from deep sea organisms on the closed-to-open conformational transition of a DNA hairpin (Hp). To this end, single-molecule Förster resonance energy transfer (smFRET) experiments were carried out in an optimized high-pressure capillary optical cell. In the absence of osmolytes, pressure shifts the conformational equilibrium of the DNA Hp towards the open, unfolded state owing to a volume decrease of about -20 cm3 mol-1. We show that the deep-sea osmolyte mixture, largely composed of TMAO, is able to rescue the DNA Hp from unfolding even up to almost 1 kbar, which is supposed to be essentially due to a distinct excluded volume effect.

Modulation of the Polymerization Kinetics of α/ß-Tubulin by Osmolytes and Macromolecular Crowding.

Tubulin is one of the main components of the cytoskeleton and can be found in nearly all eukaryotic cells. In this study, we explored the effects of kosmotropic and chaotropic osmolytes, such as trimethylamine-N-oxide (TMAO) and the metabolic waste product urea, as well as the crowding agents Ficoll and sucrose on the polymerization reaction of α/ß-tubulin. Time-dependent turbidimetry and fluorescence anisotropy experiments were performed to explore the kinetics of the polymerization reaction. Under different solvent conditions, diverse changes in the lag time, the half-life of the polymerization reaction, and the critical concentration of the polymerization reaction were observed. The apparent growth rate of the formation of microtubules was dramatically decreased in the presence of urea but significantly increased in the presence of TMAO. Measurements using mixtures of these two cosolvents showed that TMAO was able to counteract the deteriorating effect of urea on the polymerization reaction of tubulin. To create a more cell-like environment, Ficoll was added as a macromolecular crowding agent. The presence of 10 wt % Ficoll increased the apparent growth rate by one order of magnitude. Our results clearly show that the polymerization of tubulin is very sensitive to the surrounding solvent.

Cosolvent and Crowding Effects on the Temperature and Pressure Dependent Conformational Dynamics and Stability of Globular Actin.

Actin can be found in nearly all eukaryotic cells and is responsible for many different cellular functions. The polymerization process of actin has been found to be among the most pressure sensitive processes in vivo. In this study, we explored the effects of chaotropic and kosmotropic cosolvents, such as urea and the compatible osmolyte trimethylamine-N-oxide (TMAO), and, to mimic a more cell-like environment, crowding agents on the pressure and temperature stability of globular actin (G-actin). The temperature and pressure of unfolding as well as thermodynamic parameters upon unfolding, such as enthalpy and volume changes, have been determined by fluorescence spectroscopy over a wide range of temperatures and pressures, ranging from 10 to 80 °C and from 1 to 3000 bar, respectively. Complementary high-pressure NMR studies revealed additional information on the existence of native-like conformational substates of G-actin as well as a molten-globule-like state preceding the complete pressure denaturation. Different from the chaotropic agent urea, TMAO increases both the temperature and pressure stability for the protein most effectively. The Gibbs free energy differences of most of the native substates detected are not influenced significantly by TMAO. In mixtures of these osmolytes, urea counteracts the stabilizing effect of TMAO to some extent. Addition of the crowding agent Ficoll increases the temperature and pressure stability even further, thereby allowing sufficient stability of the protein at temperature and pressure conditions encountered under extreme environmental conditions on Earth.

Cosolvent and crowding effects on the polymerization kinetics of actin.

We studied the effects of kosmotropic and chaotropic cosolvents, trimethylamine-N-oxide (TMAO) and urea, as well as crowding agents (dextran) on the polymerization reaction of actin. Time-lapse fluorescence intensity and anisotropy experiments were carried out to yield information about the kinetics of the polymerization process. To also quantitatively describe the effects, cosolvents and crowding impose on the underlying rate constants of the G-to-F-transformation, an integrative stochastic simulation model was applied. Drastic and diverse changes in the lag phase and association rates as well as the critical actin concentration were observed under different solvent conditions. The association rate constant is drastically increased by TMAO but decreased by urea. In mixtures of these osmolytes, TMAO counteracts not only the deleterious effect of urea on protein structure and stability, but also on the protein-protein interactions in the course of actin polymerization. Owing to the excluded volume effect, cell-like macromolecular crowding conditions increase the nucleation and association rates by one order of magnitude. Our results clearly reveal the pronounced sensitivity of the actin polymerization reaction to changes in cosolvent conditions and the presence of macromolecular crowding, and suggest that such effects should be taken into account in any discussion of the actin polymerization reaction in vivo.