An upper limit for macromolecular crowding effects

Our studies revealed no change in protein rotational or ps-ns backbone dynamics and only mild (~0.5 kcal/mol at 37°C, pH 5.4) stabilization at a volume occupancy of 70%, which approaches the occupancy of closely packing spheres. The lack of change in rotational dynamics indicates the absence of strong crowder-protein interactions.Our observations are explained by the large size discrepancy between the protein and crowders and by the internal structure of the microgels, which provide interstitial spaces and internal pores where the protein can exist in a dilute solution-like environment. In summary, microgels that interact weakly with proteins do not strongly influence protein dynamics or stability because these large microgels constitute an upper size limit on crowding effects.The cellular interior, where most biological processes occur, is unlike the dilute solutions where most proteins are studied. The large volume excluded by high macromolecule concentrations in cells, from 20-40% [1], is predicted to change many protein properties compared to dilute solution. We used a synthetic microgel composed of poly(N-isopropylacrylamide-co-acrylic acid) [p-NIPAm-co-AAc (Figure 1A)], as a crowding agent to study the backbone dynamics and the stability of the globular test protein, chymotrypsin inhibitor 2 (CI2).p-NIPAm-co-AAc is of interest in pharmaceutical applications because it forms environmentally sensitive microgels [2]. Each microgel particle (Figure 1B) is a lightly cross-linked single polymer molecule of molecular weight 109 Da with an average of 70 monomer units between each cross link. The polymer absorbs a large amount of water resulting in spherical particles of 300 nm radii that exclude large amounts of solution volume. Their porosity arises from the balance between the external (solution) osmotic pressure and the internal osmotic pressure. This internal pressure is the result of the solvated cations that neutralize the deprotonated polymer side chains. We ch