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

Abstract

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.

In Silico Evidence That Protein Unfolding is a Precursor of Protein Aggregation

“…A sample of pioneering works in the field are. [86][87][88][89][90] The central message of these studies is that the role of the steric crowding does not significantly affect the folding. However, when globular proteins replace crowding agents, the behaviour of the system becomes difficult to explain because of the influence of protein-protein interactions.…”

Crowders and Cosolvents—Major Contributors to the Cellular Milieu and Efficient Means to Counteract Environmental Stresses

“…[119,120,149,[153][154][155][156] So far,o smolytes have been observed to increase the p m values of proteins and nucleic acids, whereas urea diminishes the pressure stability in terms of p m . [120,[154][155][156] In contrast, as the cosolvents also modulate the melting temperatureo fb iomolecules, reviewing the effect on DV u needs particularc aution owing to its temperature dependence. Generally, DV u can be obtained from PPC measurements or spectroscopic approaches.…”

“…The behavior of TMAO and urea in the vicinity of a protein was reported not to change much with pressure (from 1 to 5000 bar) and temperature (from 280 to 330 K) . High‐pressure spectroscopic approaches and pressure perturbation calorimetric (PPC) measurements revealed that both factors, the unfolding pressure ( p m ) and the volume change upon unfolding (Δ V u ), are significantly modulated . So far, osmolytes have been observed to increase the p m values of proteins and nucleic acids, whereas urea diminishes the pressure stability in terms of p m .…”

“…High‐pressure spectroscopic approaches and pressure perturbation calorimetric (PPC) measurements revealed that both factors, the unfolding pressure ( p m ) and the volume change upon unfolding (Δ V u ), are significantly modulated . So far, osmolytes have been observed to increase the p m values of proteins and nucleic acids, whereas urea diminishes the pressure stability in terms of p m . In contrast, as the cosolvents also modulate the melting temperature of biomolecules, reviewing the effect on Δ V u needs particular caution owing to its temperature dependence.…”

Temperature, Hydrostatic Pressure, and Osmolyte Effects on Liquid–Liquid Phase Separation in Protein Condensates: Physical Chemistry and Biological Implications

“…[43] Another important-yet largely unexplored-stressf actor for life's function is high hydrostatic pressure (HHP). [49][50][51][52][53][54][55][56] Al arge fraction (> 60 %) of organismsi nt he global biosphere is thriving at ad epth in excess of 1000 ma nd, therefore, exposed to pressures of 100 bar and more;a nd pressure reaches the 1kbar level at the sea floori nt he Marianat rench.H ence, HHP studies on biomolecular systems are ap rerequisite for understanding life in the deep sea, an environment whichi ss ignificant also as the potentialb irth place of life on Earth. [57,58] Further,a sathermodynamic variable, pressure can be used to perturb the intermoleculari nteraction potential between proteins and thus serve as an important physical probe for mapping their conformational landscape and phase behavior.I na ccordancew ith Le Châtelier's principle, application of increasing pressure favors states with lower partial molar volumes, hence pressure can be employed to redistribute the populations of conformational substrates by virtue of their different volumes.…”