Macromolecular Crowding In Vitro, In Vivo, and In Between

Abstract

Biochemical processes take place in heterogeneous and highly volume-occupied or crowded environments that can considerably influence the reactivity and distribution of participating macromolecules. We summarize here the thermodynamic consequences of excluded-volume and long-range nonspecific intermolecular interactions for macromolecular reactions in volume-occupied media. In addition, we summarize and compare the information content of studies of crowding in vitro and in vivo. We emphasize the importance of characterizing the behavior not only of labeled tracer macromolecules but also the composition and behavior of unlabeled macromolecules in the immediate vicinity of the tracer. Finally, we propose strategies for extending quantitative analyses of crowding in simple model systems to increasingly complex media up to and including intact cells.

Keywords: crowding theory; in-cell experiments; long-range interactions; nonspecific interactions; steric interactions.

Figure 1

Schematic depiction of volume instantaneously excluded [pink] and available (= total − excluded) [blue] to the center of mass of a newly added molecule that is small (panel A) and comparable in size (panel B) relative to the concentrated macromolecule. Excluded volume contributes RT ln (v_total/v_available) to the total free energy of interaction between the added molecule and the concentrated molecules. Figure reproduced with permission from.

Figure 2

Cartoon of the interior of an E. coli cell. Four distinct microcompartments are indicated: (1) The cytoplasm exterior to the nucleoid, consisting of primarily soluble proteins, ribonucleic acids, and macromolecular assemblies such as ribosomes and proteasomes; (2) The interior of the nucleoid, with an extremely high local concentration of DNA and DNA-binding and DNA-condensing proteins; (3) The region immediately adjacent to the inner plasma membrane, containing a high local concentration of membrane lipids and intrinsic membrane proteins, and (4) the periplasm, containing high local concentrations of membrane proteins and interstitial proteoglycans. Figure adapted from Goodsell (http://mgl.scripps.edu/people/goodsell/illustration/public).

Figure 3

Positional variability in folding kinetics and equilibria. A – Positional variation of folding relaxation time τ. blue: τ < 2.7 s, violet: 2.7s < τ < 3.2 s, red: τ > 3.2 s. Scale bar: 10 µm. Figure reproduced from with permission. B - Positional variation of melting temperature. Scale bar: 10 µm. Figure adapted from.