Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

Boqun Liu, Christoffer Åberg, Floris Jan van Eerden, Siewert Marrink, Berend Poolman, Arnold J. Boersma

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Abstract

Cells are highly crowded with proteins and polynucleotides. Any reaction that depends on the available volume can be affected by macromolecular crowding, but the effects of crowding in cells are complex and difficult to track. Here, we present a set of Forster resonance energy transfer (FRET)-based crowding-sensitive probes and investigate the role of the linker design. We investigate the sensors in vitro and in vivo and by molecular dynamics simulations. We find that in vitro all the probes can be compressed by crowding, with a magnitude that increases with the probe size, the crowder concentration, and the crowder size. We capture the role of the linker in a heuristic scaling model, and we find that compression is a function of size of the probe and volume fraction of the crowder. The FRET changes observed in Escherichia collare more complicated, where FRET increases and scaling behavior are observed solely with probes that contain the helices in the linker. The probe with the highest sensitivity to crowding in vivo yields the same macromolecularvolume fractions as previously obtained from cell dry weight. The collection of new probes provides more detailed readouts on the macromolecular crowding than a single sensor.

Original languageEnglish
Pages (from-to)1929-1939
Number of pages11
JournalBiophysical Journal
Volume112
Issue number9
DOIs
Publication statusPublished - 9-May-2017

Keywords

  • SINGLE-MOLECULE SPECTROSCOPY
  • ESCHERICHIA-COLI
  • FLUORESCENT PROTEINS
  • LIVING CELLS
  • ENERGY-TRANSFER
  • FORCE-FIELD
  • IN-VITRO
  • DEPENDENCE
  • STABILITY
  • CYTOPLASM

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