Do Macromolecular Crowding Agents Exert Only an Excluded Volume Effect? A Protein Solvation Study

Abstract

The effect of macromolecular crowding on protein structure and dynamics has mostly been explained on the basis of the excluded volume effect, its origin being entropic. In recent times a progressive shift in this view has been taking place with increasing emphasis on soft interactions that are enthalpic by nature. Using very low concentrations (1-10 g/L) of both synthetic (dextran- and poly(ethylene glycol) (PEG)-based) and protein (α-synuclein and myoglobin)-based crowders, we have shown that the solvation of probe molecule ANS (1-anilinonapthalene-8-sulfonate) bound to serum proteins bovine serum albumin (BSA) and human serum albumin (HSA) is significantly modulated in both a protein- and crowder-dependent fashion. Since under such conditions the effect of excluded volume is appreciably low, we propose that our observations are direct evidence of soft interactions between the macromolecular crowding agents used and the serum proteins. Moreover, our data reveal, that since at these low crowder concentrations major perturbations to the protein structure are unlikely to take place while minor perturbations might not be readily visible, protein solvation provides a unique spectral signature for capturing such local dynamics, thereby allowing one to decouple hard-sphere interactions from soft sphere ones. Furthermore, since fast fluctuations are known to play a major role in determining the functional characteristics of proteins and enzymes, our results suggest that such motions are prone to be modulated even when the cellular crowding conditions are quite relaxed. In other words, by the time the excluded volume effects come into the picture in the physiological milieu, modulations of functionally important protein motions that need a relatively lower activation energy have already taken place as a result of the aforementioned enthalpic (soft) interactions.

An optimized strategy to measure protein stability highlights differences between cold and hot unfolded states

“…Some studies have been performed using low molecular weight crowders whose effect can hardly be defined as crowding as it is better described as solvent perturbation56. Other authors have made use of artificial crowders such as dextran789, Ficoll101112, polyethylene glycol (PEG)913 or different proteins1415 that could potentially mimic the cellular environment. The role of soft interactions, evidenced by Pielak and co-workers414, is probably crucial inside cells, but the use of proteins as crowders may mask the true contribution of volume exclusion, the possible relationship between the dimensions of the protein under study and those of crowders and indirect effects mediated by water activity16.…”

Molecular simulations of cellular processes

“…Albeit popular, Eq. 2 and most treatments of hydrodynamic interactions do not account for the perturbation of the solvent caused by crowding agents (Harada et al 2012;Mukherjee et al 2015;Wang et al 2017b) or by protein-specific effects (Huang et al 2016). …”

Crowding in Cellular Environments at an Atomistic Level from Computer Simulations

“…7−12 However, the degree to which artificial crowders approximate real cellular environments or even reproduce just excluded-volume effects without any other artifacts is unclear. 6,13−15 At the same time, increasing attention is now focused on the effects of interactions between the cellular components 16−24 and altered solvent properties, 25−30 often contributing enthalpically, 15,31,32 as well as the consequences of a substantial compositional heterogeneity present in biological cells. 33,34 …”