Design and Properties of Genetically Encoded Probes for Sensing Macromolecular Crowding

Biophysical Journal
Boqun LiuArnold J Boersma

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 Förster 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 coli are 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 macromolecular volume 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.

Citations

May 19, 2017·The Journal of Physical Chemistry. B·Megan CurrieAhmed A Heikal
Dec 1, 2017·Biophysics Reviews·Fabio Trovato, Giordano Fumagalli
Sep 12, 2018·Physical Chemistry Chemical Physics : PCCP·Hong Bok LeeAhmed A Heikal
Mar 15, 2019·Nanoscale·Christopher G ClarksonMark Geoghegan
Mar 6, 2019·Journal of Bacteriology·Boqun LiuArnold J Boersma
Aug 6, 2019·Chembiochem : a European Journal of Chemical Biology·Hendrik R SikkemaBert Poolman
Jan 15, 2019·Molecular Biology of the Cell·T J Mitchison
Oct 30, 2020·Microbiology and Molecular Biology Reviews : MMBR·Alexander Cambré, Abram Aertsen
Dec 10, 2020·Trends in Biotechnology·Alexander Gräwe, Viktor Stein
Aug 31, 2017·ACS Chemical Biology·Boqun LiuArnold J Boersma
Jan 10, 2019·Analytical Chemistry·Shuntaro TakahashiNaoki Sugimoto
May 7, 2019·The Journal of Physical Chemistry. B·Niklas O JunkerJörg Fitter
Nov 17, 2020·The Journal of Physical Chemistry Letters·David MosesShahar Sukenik

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