Associative Interactions in Crowded Solutions of Biopolymers Counteract Depletion Effects

Abstract

The cytosol of Escherichia coli is an extremely crowded environment, containing high concentrations of biopolymers which occupy 20-30% of the available volume. Such conditions are expected to yield depletion forces, which strongly promote macromolecular complexation. However, crowded macromolecule solutions, like the cytosol, are very prone to nonspecific associative interactions that can potentially counteract depletion. It remains unclear how the cytosol balances these opposing interactions. We used a FRET-based probe to systematically study depletion in vitro in different crowded environments, including a cytosolic mimic, E. coli lysate. We also studied bundle formation of FtsZ protofilaments under identical crowded conditions as a probe for depletion interactions at much larger overlap volumes of the probe molecule. The FRET probe showed a more compact conformation in synthetic crowding agents, suggesting strong depletion interactions. However, depletion was completely negated in cell lysate and other protein crowding agents, where the FRET probe even occupied slightly more volume. In contrast, bundle formation of FtsZ protofilaments proceeded as readily in E. coli lysate and other protein solutions as in synthetic crowding agents. Our experimental results and model suggest that, in crowded biopolymer solutions, associative interactions counterbalance depletion forces for small macromolecules. Furthermore, the net effects of macromolecular crowding will be dependent on both the size of the macromolecule and its associative interactions with the crowded background.

Identification of primary and secondary UBA footprints on the surface of ubiquitin in cell‐mimicking crowded solution

“…Volume occupancy by Ficoll was calculated from a reported partial specific volume of 650 cm 3 ·g −1 , a value in agreement with our experimental verification of volume change upon dissolution of a known mass of solute. The volume fraction occupied by the crowder, ϕ C , in a 200 g·L −1 Ficoll solution was determined to be 13%, assuming no volume changes resulted from perturbations of solvent molecules.…”

Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

“…The incorporation of paired lysines and glutamates would prevent interactions, allowing steric effects to govern the conformation. In general, the observation that in-cell behavior is different compared to in vitro crowding is not very surprising: chemical nonspecific interactions seem to dominate over the steric crowding for most reported small proteins (6)(7)(8)(9)(10)(11)(12). Hence, this is the most likely explanation, and it is remarkable that the steric compression appears to be regained by the presence of these helices.…”

“…The probes expanded in the presence of small amounts (1% wt/wt) of g-Globulins, which suggests that g-Globulins bind the probes. The probes did not expand further by addition of 10% wt/wt g-Globulins, which could be due to saturation of binding sites, balancing excluded volume effects (9), or the decrease of attractive interactions of concentrated antibodies (37,38). We observed compression of the three probes in the presence of a variety of macromolecular crowders based on the carbohydrates Ficoll 70 and 400 kDa, Dextran 40 and 6 kDa, and the proteins BSA and ovalbumin, all at 10% wt/wt (Fig.…”

“…The entropic penalty can be relieved by reducing the volume of the introduced protein. In the cell, other interactions are able to attenuate this entropic effect, resulting in net effects that are often different from what would be predicted solely due to steric exclusion (5)(6)(7)(8)(9)(10)(11)(12). This makes that crowding effects are unpredictable in cells, and can be overshadowed by other nonspecific interactions if the excluded volume effects are small.…”

Macromolecular Crowding In Vitro , In Vivo , and In Between

“…Excluded volume theory predicts that the contribution of excluded volume to a particular reaction equilibrium will only become substantial when the size of the probe is comparable to or exceeds that of the major crowding species 13 and when the probe reaction (isomerization or association) is accompanied by a substantial reduction of the ratio of solvent-exposed surface to volume 12 . These predictions are borne out by the following observations: (1) The formation of polymers of sickle hemoglobin (HbS) is facilitated by high concentrations of bovine serum albumin and hemoglobin mutants that do not coprecipitate with HbS 90 ; (2) the formation of amyloid fibers of alpha synuclein is strongly promoted by high concentrations of other proteins as well as by nonionic polymers 91 ; and (3) the formation of fiber bundles of FtsZ is strongly facilitated not only by the addition of traditional polymeric crowding agents, but also by the addition of 10 % (by volume) of BSA, ovomucoid, and Escherichia coli lysate 92 .…”