Abstract
BackgroundSolutions containing high macromolecule concentrations are predicted to affect a number of protein properties compared to those properties in dilute solution. In cells, these macromolecular crowders have a large range of sizes and can occupy 30% or more of the available volume. We chose to study the stability and ps-ns internal dynamics of a globular protein whose radius is ~2 nm when crowded by a synthetic microgel composed of poly(N-isopropylacrylamide-co-acrylic acid) with particle radii of ~300 nm.ResultsOur 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.ConclusionsOur 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.
Highlights
Solutions containing high macromolecule concentrations are predicted to affect a number of protein properties compared to those properties in 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)
Experiments were performed by using samples comprising 1 mM CI2 in 50 mM sodium acetate solution, pH 5.4 at 37°C
Summary
Solutions containing high macromolecule concentrations are predicted to affect a number of protein properties compared to those properties in dilute solution. We chose to study the stability and ps-ns internal dynamics of a globular protein whose radius is ~2 nm when crowded by a synthetic microgel composed of poly(N-isopropylacrylamide-co-acrylic acid) with particle radii of ~300 nm. 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). 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. We chose this crowding agent because its status as a drug delivery molecule makes it pharmaceutically relevant, and its ability to take up water provides a model for volume exclusion by a molecule much larger than our test protein
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