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Crowding and Confinement Effects on Protein Diffusion In Vivo

Departments of Chemistry,¹ Biochemistry, University of Wisconsin—Madison, Madison, Wisconsin 53706²

Received 27 December 2005/ Accepted 13 April 2006

The first in vivo measurements of a protein diffusion coefficient versus cytoplasmic biopolymer volume fraction are presented. Fluorescence recovery after photobleaching yields the effective diffusion coefficient on a 1-µm-length scale of green fluorescent protein within the cytoplasm of Escherichia coli grown in rich medium. Resuspension into hyperosmotic buffer lacking K⁺ and nutrients extracts cytoplasmic water, systematically increasing mean biopolymer volume fraction, <>, and thus the severity of possible crowding, binding, and confinement effects. For resuspension in isosmotic buffer (osmotic upshift, or , of 0), the mean diffusion coefficient, <D>, in cytoplasm (6.1 ± 2.4 µm² s^–1) is only 0.07 of the in vitro value (87 µm² s^–1); the relative dispersion among cells, _D/<D> (standard deviation, _D, relative to the mean), is 0.39. Both <D> and _D/<D> remain remarkably constant over the range of values of 0 to 0.28 osmolal. For a value of 0.28 osmolal, formation of visible plasmolysis spaces (VPSs) coincides with the onset of a rapid decrease in <D> by a factor of 380 over the range of values of 0.28 to 0.70 osmolal and a substantial increase in _D/<D>. Individual values of D vary by a factor of 9 x 10⁴ but correlate well with f_VPS, the fractional change in cytoplasmic volume on VPS formation. The analysis reveals two levels of dispersion in D among cells: moderate dispersion at low values for cells lacking a VPS, perhaps related to variation in or biopolymer organization during the cell cycle, and stronger dispersion at high values related to variation in f_VPS. Crowding effects alone cannot explain the data, nor do these data alone distinguish crowding from possible binding or confinement effects within a cytoplasmic meshwork.

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