Diffusion NMR and Rheology of a Model Polymer in Bacterial Cell Lysate Crowders

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The intracellular milieu is crowded and heterogeneous, and this can have profound consequences for biomolecule motions and biochemical kinetics. Macromolecular crowding has been traditionally studied in artificial crowders like Ficoll and dextran or globular proteins such as bovine serum albumin. It is, however, not clear if the effects of artificial crowders on such phenomena are the same as the crowding that is experienced in a heterogeneous biological environment. Bacterial cells, for example, are composed of heterogeneous biomolecules with different sizes, shapes, and charges. Using crowders composed of one of three different pretreatments of bacterial cell lysate (unmanipulated, ultracentrifuged, and anion exchanged), we examine the effects of crowding on the diffusivity of a model polymer. We measure the translational diffusivity, via diffusion NMR, of the test polymer polyethylene glycol (PEG) in these bacterial cell lysates. We show that the small (R_g ∼ 5 nm) test polymer shows a modest decrease in self-diffusivity with increasing crowder concentration for all lysate treatments. The corresponding self-diffusivity decrease in the artificial Ficoll crowder is much more pronounced. Moreover, a comparison of the rheological response of biological and artificial crowders shows that while the artificial crowder Ficoll exhibits a Newtonian response even at high concentrations, the bacterial cell lysate is markedly non-Newtonian; it behaves like a shear-thinning fluid with a yield stress. While at any concentration the rheological properties are sensitive to both lysate pretreatment and batch-to-batch variations, the PEG diffusivity is nearly unaffected by the type of lysate pretreatment.