We studied the effects of temperature and hydrostatic pressure on the dynamical properties and folding stability of highly concentrated lysozyme solutions in the absence and presence of the osmolytes trimethylamine-N-oxide (TMAO) and urea. Elastic incoherent neutron scattering (EINS) was applied to determine the mean-squared displacement (MSD) of the protein's hydrogen atoms to yield insights into the effects of these cosolvents on the averaged sub-nanosecond dynamics in the pressure range from ambient up to 4000 bar. To evaluate the additional effect of self-crowding, two protein concentrations (80 and 160 mg mL) were used. We observed a distinct effect of TMAO on the internal hydrogen dynamics, namely a reduced mobility. Urea, on the other hand, revealed no marked effect and consequently, no counteracting effect in an urea-TMAO mixture was observed. Different from the less concentrated protein solution, no significant effect of pressure on the MSD was observed for 160 mg mL lysozyme. The EINS experiments were complemented by Fourier-transform infrared (FTIR) spectroscopy measurements, which led to additional insights into the folding stability of lysozyme under the various environmental conditions. We observed a stabilization of the protein in the presence of the compatible osmolyte TMAO and a destabilization in the presence of urea against temperature and pressure for both protein concentrations. Additionally, we noticed a slight destabilizing effect upon self-crowding at very high protein concentration (160 mg mL), which is attributable to transient destabilizing intermolecular interactions. Furthermore, a pressure-temperature diagram could be obtained for lysozyme at these high protein concentrations that mimics densely packed intracellular conditions.

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.

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.

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/β-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.

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