Effects of macromolecular crowding on protein folding and aggregation

Abstract

We have studied the effects of polysaccharide and protein crowding agents on the refolding of oxidized and reduced hen lysozyme in order to test the prediction that association constants of interacting macromolecules in living cells are greatly increased by macromolecular crowding relative to their values in dilute solutions. We demonstrate that whereas refolding of oxidized lysozyme is hardly affected by crowding, correct refolding of the reduced protein is essentially abolished due to aggregation at high concentrations of crowding agents. The results show that the protein folding catalyst protein disulfide isomerase is particularly effective in preventing lysozyme aggregation under crowded conditions, suggesting that crowding enhances its chaperone activity. Our findings suggest that the effects of macromolecular crowding could have major implications for our understanding of how protein folding occurs inside cells.

Molecular crowding enhances native structure and stability of α/β protein flavodoxin

“…Earlier experimental work has focused on the effects of crowding on kinetic refolding of complex proteins and on protein nonnative states at extreme conditions, whereas theoretical approaches have instead involved simple lattice models or small peptide systems (1,(9)(10)(11)(12)). …”

“…This means that a significant fraction of the intracellular space is not available to other macromolecular species. It has been estimated that the concentration of macromolecules in the cytoplasm is in the range of 80-400 mg/ml (1,2). All macromolecules in physiological fluids collectively occupy between 10% and 40% of the total fluid volume (3,4).…”

“…Experimental and theoretical work has demonstrated large effects of crowding on the thermodynamics and kinetics of many biological processes, including protein binding, folding, and aggregation (1,(9)(10)(11)(12).…”

“…For example, the effects of crowding agents on the refolding of reduced denatured lysozyme (1,9), oligomerization of GroEL subunits (13), self-assembly of the cell division protein FtsZ (14) and the capsid protein of HIV (15), and amyloid formation of the human apolipoprotein C-II (16) have been reported. A few studies have focused on the ability of crowding agents to induce conformational changes in unfolded states of proteins (17,18).…”

Self-crowding induced phase separation in protein dispersions

“…This implies that in crowded environments, where the free volume is limited due to the presence of a high concentration of macromolecules, such as is the case in the cell, 7,8 interactions between proteins may be different from that in a dilute solution. [9][10][11][12][13] Indeed, it is well known that crowding tends to increase protein complexation rates and shifts binding equilibria towards assembled states in order to increase the free volume available in the solution. 9 Crowding stabilises the compact (native) protein conformation for the same reason.…”