Molecular Crowding: The History and Development of a Scientific Paradigm

Abstract

It is now generally accepted that macromolecules do not act in isolation but "live" in a crowded environment, that is, an environment populated by numerous different molecules. The field of molecular crowding has its origins in the far 80s but became accepted only by the end of the 90s. In the present issue, we discuss various aspects that are influenced by crowding and need to consider its effects. This Review is meant as an introduction to the theme and an analysis of the evolution of the crowding concept through time from colloidal and polymer physics to a more biological perspective. We introduce themes that will be more thoroughly treated in other Reviews of the present issue. In our intentions, each Review may stand by itself, but the complete collection has the aspiration to provide different but complementary perspectives to propose a more holistic view of molecular crowding.

Figure 1

Top: CP-mixture in the colloid limit (R_g < R) with macromolecules as crowder (left), R_g∼ R (center), CP-mixture in the protein limit (R_g> R) with colloid-like proteins as crowder (right). Bottom: dimensions of colloids and macromolecules (left), colloids in semidilute solution of macromolecules with macromolecules as crowder (center), correlation length ξ (right).

Figure 2

Workflow to understand biomolecular reactions in biological environments with increasing complexity.

Figure 3

Excluded volume effect favors the more compact conformations of proteins due to hard-core repulsions. The folded native state of a protein is favored over the expanded denatured state because of its compact structure. The excluded volume effect increases the free energy of both, the folded and the unfolded state. However, the increase in free energy is larger for the expanded unfolded state leading to an overall stabilization of the native protein.

Figure 4

Folding equilibrium of ubiquitin in dilute and crowded solution. The native state of ubiquitin is stabilized relative to the denatured state via an enthalpic mechanism.

Figure 5

Diagram summarizes the effects of molecular crowding on protein stability and/or on protein ability to interact giving rise to association phenomena leading to different supramolecular structures. Protein aggregation is a complex, often hierarchical, multistep process determined by the interconnection and modulation of multiple mechanisms appearing in different time and length scales. The presence of crowders may impact protein aggregation at different levels involving a complex interplay of various effects, such as excluded volume, changes in solution viscosity, modification of dominant short- and long-range interactions and water ordering.

Figure 6

Schematic overview of the effects of molecular crowding (center, green spheres) on LLPS and biomolecular condensates formed by partially disordered proteins (center, blue). Crowding generally promotes LLPS, which is quantified by a shift of the LLPS phase boundary, and the addition of crowding agents can induce LLPS. Crowding agents may also change physicochemical properties of BMCs after formation, including composition (via partitioning), density, viscosity and surface tension (wetting). The presence of crowders can influence the size distribution of BMCs via altered nucleation and coalescence rates. Finally, crowders can induce or speed up aging of BMCs, which may result in kinetically arrested gel-like states and aberrant phase transitions.

Figure 7

Mussel byssal plaque’s formation and deposition. On the left, from the top to the bottom: Mussel generates several byssal threads ending with plaques firmly attached to the surface. Below follows schematic anatomic representation of byssal threads, from the stem where the threads’ elongation starts, to the core formed inside the elongated thread, protected by a cuticle and the adhesive plaque. On the right, from the top to the bottom: schematic representation of the different steps for the plaque formation and deposition. Mussel foot anchors on the surface and creates a cavitation, known also as distal depression, which allows to maintain different chemical conditions than those of the outer environment. Under acidic pH, low ionic strength and controlled redox conditions, the mussel foot proteins responsible of the adhesion process (Pvfp-5β, Pvfp3α, and Pvfp-6) are secreted and undergo coacervation to enhance spreading and wettability on the surface. Finally, immediately after the foot is released the plaque undergoes solidification in contact with seawater and firmly remains anchored to the surface.