Excluded Volume Effects Inside the Living Cell

Abstract

Proteins and nucleic acids fold and behave in a highly occupied matrix of macromolecules, the cellular environment. Although this environment is filled up to a volume of 40% with macromolecules, the effect of crowding on biochemical reactions as well as biophysical properties of proteins has been rarely considered. Therefore, studies on macromolecular crowding are mainly conducted using artificially created polymer based substances as mimics of the in cell environment. Still, a common concept how the excluded volume effect in the cell affects protein folding, function and structure is lacking. Understanding these properties will be important to unravel the underlying mechanism of protein misfolding and aggregation as well as the behavior of intrinsically disordered proteins in a living cell.We introduce a FRET based random coil polymer to characterize crowding both in vitro and inside a living cell. We find different compression of the polymer in artificial crowding substances compared to protein crowders such as BSA or oocyte lysate. Injection of the probe inside cells confirms this result and reveals a heterogeneous environment which, on average, shows comparable polymer conformations as in diluted buffer. The polymer conformation is used to quantify crowding differences in the cytosol and the nucleus as well as to identify crowding in cells influenced by different extrinsic conditions. Severe osmotic stress is used to probe compression of the polymer in the highly concentrated cytosolic environment.We conclude that the FRET labelled polymer provides a new approach to investigate and characterize the cellular solvation properties with high spatio-temporal resolution in a variety of systems and identify crowding differences due to the architecture of the cellular matrix. Therefore, it will help to understand how the cell, as the native environment in which proteins evolved, might modulate and tune biomolecule properties and functions.