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ENZYMES AND PROTEINS

Crowding and Confinement Effects on Protein Diffusion In Vivo

Michael C. Konopka, Irina A. Shkel, Scott Cayley, M. Thomas Record, James C. Weisshaar
Michael C. Konopka
1Departments of Chemistry
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Irina A. Shkel
1Departments of Chemistry
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Scott Cayley
1Departments of Chemistry
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M. Thomas Record
1Departments of Chemistry
2Biochemistry, University of Wisconsin—Madison, Madison, Wisconsin 53706
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James C. Weisshaar
1Departments of Chemistry
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  • For correspondence: weisshaar@chem.wisc.edu
DOI: 10.1128/JB.01982-05
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ABSTRACT

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, <φ>, and thus the severity of possible crowding, binding, and confinement effects. For resuspension in isosmotic buffer (osmotic upshift, or Δ, 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, σD/<D> (standard deviation, σD, relative to the mean), is 0.39. Both <D> and σD/<D> remain remarkably constant over the range of Δ values of 0 to 0.28 osmolal. For a Δ 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 Δ values of 0.28 to 0.70 osmolal and a substantial increase in σD/<D>. Individual values of D vary by a factor of 9 × 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 Δ values for cells lacking a VPS, perhaps related to variation in φ or biopolymer organization during the cell cycle, and stronger dispersion at high Δ 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.

  • Copyright © 2006 American Society for Microbiology
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Crowding and Confinement Effects on Protein Diffusion In Vivo
Michael C. Konopka, Irina A. Shkel, Scott Cayley, M. Thomas Record, James C. Weisshaar
Journal of Bacteriology Aug 2006, 188 (17) 6115-6123; DOI: 10.1128/JB.01982-05

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Crowding and Confinement Effects on Protein Diffusion In Vivo
Michael C. Konopka, Irina A. Shkel, Scott Cayley, M. Thomas Record, James C. Weisshaar
Journal of Bacteriology Aug 2006, 188 (17) 6115-6123; DOI: 10.1128/JB.01982-05
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KEYWORDS

Escherichia coli
Green Fluorescent Proteins

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