24.11 Macromolecular Crowding and Internal Rheology

Macromolecular Crowding and Internal Rheology refers to the physical consequences of high concentrations of large molecules occupying a synthetic cell's confined internal volume, encompassing how this crowding affects molecular behavior, reaction rates, and diffusion, along with the internal flow and deformation properties that emerge from this densely packed environment.

Quantifying Crowding

Synthetic Cell Macromolecular Crowding

Macromolecular crowding refers to the condition in which a substantial fraction of a synthetic cell's internal volume is occupied by large molecules, producing physical effects on the behavior of other molecules that would not occur in a more dilute solution.

Internal Macromolecular Volume Fraction

The volume fraction occupied by macromolecules provides a direct, quantifiable measure of crowding intensity, describing what proportion of the internal space is physically excluded from occupation by any other molecule at a given moment.

Contributors to Crowding

Protein, Nucleic Acid, and Synthetic Polymer Crowding Contribution

Proteins contribute substantially to overall crowding through their sheer abundance and size, nucleic acids contribute their own significant volume, particularly where the genome or other genetic material occupies considerable internal space, and any synthetic polymers incorporated into the cell design add a further, engineered contribution to total crowding.

Ribosome Crowding Contribution

Ribosomes, being both numerous and comparatively large, often represent a disproportionately significant contributor to overall macromolecular crowding relative to their individual molecular count.

Functional Consequences

Molecular Exclusion and Association Enhancement

Crowding-induced molecular exclusion physically restricts the volume available to other molecules, effectively increasing their local concentration, while crowding-induced association enhancement increases the tendency of molecules to bind one another due to this same volume exclusion effect.

Aggregation Risk and Reaction Rate Change

Crowding-induced aggregation risk reflects the increased likelihood that proteins or other macromolecules will inappropriately clump together under crowded conditions, while crowding-induced reaction rate change describes how the effective concentration increase from crowding alters the speed of biochemical reactions relative to dilute conditions.

Diffusion Reduction

Crowding-induced diffusion reduction slows the movement of molecules through the internal environment, since dense macromolecular occupation physically impedes free movement compared to a less crowded medium.

Flow and Deformation Properties

Internal Viscosity Regulation and Viscoelasticity

Internal viscosity regulation manages the overall resistance of the crowded interior to molecular movement, while internal viscoelasticity describes the combined viscous and elastic behavior of this dense internal environment, reflecting properties intermediate between a simple liquid and a solid.

Local Rheological Heterogeneity

Local rheological heterogeneity acknowledges that crowding and its associated flow properties are not necessarily uniform throughout the synthetic cell, with some regions potentially more densely packed or more resistant to flow than others.

Crowding Changes Over Time

Redistribution during Volume Change, Gene Expression, and Genome Replication

Crowding redistributes as cell volume changes, since a fixed quantity of macromolecules occupies a different volume fraction as the cell expands or contracts, and similarly redistributes during gene expression, as new protein and RNA molecules are produced, and during genome replication, as genetic material duplicates within the same confined space.

Managing Crowding Levels

Concentration Relief and Dilution Compensation

Macromolecular concentration relief mechanisms reduce excessive crowding by removing or redistributing macromolecules, while macromolecular dilution compensation addresses the opposite problem, restoring functional crowding levels when growth or other processes have diluted macromolecular concentration below its intended range.

Synthetic Cell Crowding Stability Limit

The overall crowding stability limit reflects the range of macromolecular volume fraction within which the synthetic cell's reaction rates, diffusion, and structural integrity remain compatible with proper function, beyond which excessive or insufficient crowding begins to disrupt normal cellular processes.

Summary

Macromolecular Crowding and Internal Rheology encompasses the volume fraction occupied by proteins, nucleic acids, synthetic polymers, and ribosomes, and the resulting effects on molecular exclusion, association, aggregation, reaction rates, and diffusion. Managing internal viscosity, viscoelasticity, and crowding redistribution during volume change and macromolecular synthesis determines the overall crowding stability limit within which a synthetic cell's internal environment remains functional.