Influence of cosolvents, self-crowding, temperature and pressure on the sub-nanosecond dynamics and folding stability of lysozyme

Abstract

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.

Stability of the chaperonin system GroEL–GroES under extreme environmental conditions

“…Differently, marked stabilizing effects of some monomeric proteins (e.g., SNase, RNase A) by TMAO against pressureinduced unfolding and favorable effects of TMAO on enzyme function, protein polymerization and channel activity under high-pressure stress have been reported. 41,[56][57][58][59][60][61][62][63] The minor (possibly stabilizing) effect observed here is probably due to a smaller degree of unfolding and exposure of solvent accessible surface area (SASA), rendering the excluded volume effect imposed by TMAO less effective.…”

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

“…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 .…”

“…This means that TMAO may act as a chemical chaperone to assist the rather pressure‐sensitive de novo formation of actin filaments in deep‐sea organisms. Furthermore, TMAO is able to reduce the sub‐nanosecond dynamics of the protein's hydrogen atoms, presumably via water structure enhancement . Generally, the protein dynamics is supposed to depend on the water dynamics, whereby internal protein motions are mainly slaved to the dynamics of the first hydration shell …”

“…[68] High-pressure spectroscopic approaches and pressure perturbation calorimetric (PPC) measurements revealed that both factors, the unfolding pressure (p m )a nd the volume change upon unfolding (DV u ), are significantly modulat- ChemPhysChem 2017ChemPhysChem , 18,2951ChemPhysChem -2972 www.chemphyschem.org ed. [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.…”

“…[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.…”