Macromolecular Crowding: How Shape and Interactions Affect Diffusion.

A significant fraction of the cell volume is occupied by various proteins, polysaccharides, nucleic acids, etc., which considerably reduces the mobility of macromolecules. Theoretical and experimental work so far have mainly focused on the dependence of the mobility on the occupied volume, while the effect of a macromolecular shape received less attention. Herein, using fluorescence correlation spectroscopy (FCS) and Brownian dynamics (BD) simulations, we report on a dramatic slowdown of tracer diffusion by cylindrically shaped double-stranded (ds) DNAs (16 nm in length). We find, for instance, that the translational diffusion coefficient of a streptavidin tracer is reduced by about 60% for a volume fraction of dsDNA as low as just 5%. For comparison, for a spherical crowder (Ficoll70) the slowdown is only 10% at the same volume fraction and 60% reduction occurs at a volume fraction as high as 35%. BD simulations reveal that this reduction can be attributed to a larger volume excluded to a tracer by dsDNA particles, as compared with spherical Ficoll70 at the same volume fraction, and to the differences in the tracer-crowder attractive interactions. In addition, we find using BD simulations that rotational diffusion of dsDNA is less affected by the crowder shape than its translational motion. Our results show that diffusion in crowded systems is determined not merely by the occupied volume fraction, but that the shape and interactions can determine diffusion, which is relevant to the diverse intracellular environments inside living cells.

Impact of Molecular Crowding on Translational Mobility and Conformational Properties of Biological Macromolecules.

Effects of molecular crowding on structural and dynamical properties of biological macromolecules do depend on the concentration of crowding agents but also on the molecular mass and the structural compactness of the crowder molecules. By employing fluorescence correlation spectroscopy (FCS), we investigated the translational mobility of several biological macromolecules ranging from 17 kDa to 2.7 MDa. Polyethylene glycol and Ficoll polymers of different molecular masses were used in buffer solutions to mimic a crowded environment. The reduction in translational mobility of the biological tracer molecules was analyzed as a function of crowder volume fractions and was generally more pronounced in PEG as compared to Ficoll solutions. For several crowding conditions, we observed a molecular sieving effect, in which the diffusion coefficient of larger tracer molecules is reduced to a larger extent than predicted by the Stokes-Einstein relation. By employing a FRET-based biosensor, we also showed that a multiprotein complex is significantly compacted in the presence of macromolecular crowders. Importantly, with respect to sensor in vivo applications, ligand concentration determining sensors would need a crowding specific calibration in order to deliver correct cytosolic ligand concentration.