Macromolecular crowding perturbs protein refolding kinetics: implications for folding inside the cell

Abstract

We have studied the effects of macromolecular crowding on protein folding kinetics by studying the oxidative refolding of hen lysozyme in the absence and presence of high concentrations of bovine serum albumin and Ficoll 70. The heterogeneity characteristic of the lysozyme refolding process is preserved under crowded conditions. This, together with the observation that the refolding intermediates that accumulate to significant levels are very similar in the absence and presence of Ficoll, suggests that crowding does not alter substantially the energetics of the protein folding reaction. However, the presence of high concentrations of macromolecules results in the acceleration of the fast track of the refolding process whereas the slow track is substantially retarded. The results can be explained by preferential excluded volume stabilization of compact states relative to more unfolded states, and suggest that, relative to dilute solutions, the rates of many protein folding processes are likely to be altered under conditions that more closely resemble the intracellular environment.

Firefly luciferase mutants as sensors of proteome stress

“…It has been estimated that macromolecules occupy around 20-30% of the total cell's volume and therefore this fraction is physically unavailable to other molecules. This results in exclusion of volume that a molecule can occupy and causes crowding (van den Berg et al, 2000). Macromolecular crowding also plays a role in the functional interactions between molecules (Martin and Hartl, 1997).…”

Structure of phase-separated athermal colloid-polymer systems in the protein limit

“…These systems are of particular interest because they serve as a convenient model to study depletion interactions, which also play a considerable role in biological settings, contributing to protein folding, fiber bundling, and the formation of supramolecules [2][3][4][5]. Depletion arises as two or more confining surfaces approach one another and exclude a region between them where the depletant, in this case polymer, can no longer exist.…”

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

“…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).…”