Polyethylene Glycol Crowder’s Effect on Enzyme Aggregation, Thermal Stability, and Residual Catalytic Activity

Abstract

Protein stability and performance in various natural and artificial systems incorporating many other macromolecules for therapeutic, diagnostic, sensor, and biotechnological applications attract increasing interest with the expansion of these technologies.Here we address the catalytic activity of lysozyme protein (LYZ) in the presence of a polyethylene glycol (PEG) crowder in a broad range of concentrations and temperatures in aqueous solutions of two different molecular mass PEG samples (M w = 3350 and 10000 g/mol). The phase behavior of PEG−protein solutions is examined by using dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS), while the enzyme denaturing is monitored by using an activity assay (AS) and circular dichroism (CD) spectroscopy. Molecular dynamic (MD) simulations are used to illustrate the effect of PEG concentration on protein stability at high temperatures. The results demonstrate that LYZ residual activity after 1 h incubation at 80 °C is improved from 15% up to 55% with the addition of PEG. The improvement is attributed to two underlying mechanisms. (i) Primarily, the stabilizing effect is due to the suppression of the enzyme aggregation because of the stronger PEG−protein interactions caused by the increased hydrophobicity of PEG and lysozyme at elevated temperatures. (ii) The MD simulations showed that the addition of PEG to some degree stabilizes the secondary structures of the enzyme by delaying unfolding at elevated temperatures. The more pronounced effect is observed with an increase in PEG concentration. This trend is consistent with CD and AS experimental results, where the thermal stability is strengthened with increasing of PEG concentration and molecular mass. The results show that the highest stabilizing effect is approached at the critical overlap concentration of PEG.

Atomistic Simulation of Lysozyme in Solutions Crowded by Tetraethylene Glycol: Force Field Dependence

“…Note that both force fields predicted a slightly larger fraction of β -sheet structure. Because the formation of β -sheet was argued to increase the possibility of protein aggregation [ 6 , 83 , 84 ], both force fields should be used with caution in simulations of concentrated systems crowded by proteins. Note that no significant changes were observed in the protein secondary structures for all of the simulated systems ( Table S6 ).…”

“…Note that no significant changes were observed in the protein secondary structures for all of the simulated systems ( Table S6 ). Increasing the temperature to, for instance, 500 K, Wang and coworkers observed dramatic changes in the lysozyme conformation [ 6 ]. The experimental melting point of lysozyme was roughly 360 K [ 2 , 82 ], and we did not try to simulate the protein at a high temperature over 370 K because the unfolded protein did not allow a meaningful and direct comparison of protein diffusion in different crowed environments (discussed in the following; Section 2.4 ).…”

“…The concentration of macromolecular crowders goes up to 400 g/L, and these crowders occupy 5–40% of the total volume physically [ 1 ]. The resulting excluded volume affects the stability and folding of proteins [ 2 , 3 , 4 , 5 ] as well as enzyme activity [ 6 , 7 , 8 ] via a reduced diffusion and collision [ 9 ] between the molecules. Besides such “hard” interactions (steric, entropic effects), “soft” interactions (transient, enthalpic effects) contribute significantly to the structural, dynamic, and thermodynamic properties of biomolecules in crowded environments as well [ 10 , 11 , 12 ].…”

“…A number of factors such as shape, size, concentration, and composition of molecular crowders were reported to be influential [ 18 , 24 , 25 ]. Polyethylene glycol (PEG) was often used as a crowder to mimic the cell-like environment [ 6 , 26 , 27 , 28 , 29 , 30 ]. For instance, Nolan and coworkers reported a noticeable increase in the thermal stability and catalytic activity of β -galactosidase in the presentence of PEG with a concentration of 25% and 35% ( w/v ) [ 29 ].…”

“…For instance, Nolan and coworkers reported a noticeable increase in the thermal stability and catalytic activity of β -galactosidase in the presentence of PEG with a concentration of 25% and 35% ( w/v ) [ 29 ]. Wang and coworkers indicated that the increase in the residual activity of lysozyme was ascribed to the fact that the addition of PEG suppressed enzyme aggregation via a strong PEG–protein interaction and stabilized the protein secondary structure somewhat [ 6 ]. On the contrary, soft interactions with PEG were also observed to induce structural changes and destabilize proteins [ 28 , 31 , 32 , 33 , 34 ].…”