title: Protein diffusion in the cytoplasm of Escherichia coli
title: Proteindiffusion im Zytoplasma von Escherichia coli
titleType: AlternativeTitle
Creators: name: Bellotto, Nicola
Publisher: name: Philipps-Universität Marburg
Year: 2022
Subjects: subject: Fluoreszenzkorrelationsspektroskopie
subject: Biophysics
subject: Fluorescence correlation spectroscopy
subject: Protein diffusion
subject: GFP
subject: Life sciences
subject: Biowissenschaften, Biologie
subject: GFP;
subject: Cytoplasm
subject: Zytoplasma
subject: Brownian diffusion
subject: Macromolecular
subject: Proteindiffusion
subject: Mikrobiologie
subject: Microbiology
subject: FOS: Biological sciences
subject: Brownsche Diffusion
subject: Biophysik

description: Inside prokaryotic cells, passive translational diffusion is fundamental to most cellular processes because it allows the sorting of macromolecules, proteins in particular, and it sets the upper limit for biochemical reactions to happen. In the highly crowded, confined, and non-homogeneous environment of a bacterial cell, protein diffusion is typically hindered. Drawing general conclusions about the size dependence of protein diffusion from previous studies was hampered by the limited number of protein probes investigated and by the differences in the strain, the growth conditions, or the measurement technique. Furthermore, while the impact of several physiological perturbations on protein diffusion has been established, most of the previous studies used either large particles or free GFP as probes. How these perturbations affect the diffusional properties of the cytoplasm over the entire physiological range of protein sizes remains unknown. Here, we address these limitations by systematically analyzing the mobility of a large number of differently-sized cytoplasmic fluorescent protein constructs in the cytoplasm of Escherichia coli by fluorescence correlation spectroscopy (FCS) under standardized conditions. By combining experimental evidences with simulations and theoretical modeling, we concluded that protein mobility in the cytoplasm could be well described by Brownian diffusion in the confined geometry of the bacterial cell and at the high viscosity imposed by macromolecular crowding. Pronounced sub-diffusion and hindered mobility were only observed for proteins with extensive interactions within the cytoplasm. We observed that the size dependence of protein diffusion for the majority of tested proteins, whether native or foreign to E. coli, it was well consistent with the Stokes-Einstein relation once the specific dumbbell shape of protein fusions is taken into account. Furthermore, such size dependence was conserved over the different spatiotemporal scales explored by FCS and fluorescence recovery after photobleaching (FRAP). A subset of constructs, spanning over a wide range of physiologically relevant protein masses, was then used to probe the effect on diffusion of diverse, biologically meaningful, physicochemical perturbations and of cell growth. In particular, protein diffusion became markedly faster in actively growing cells, at high temperature, or upon treatment with rifampicin, and slower at high osmolarity. Importantly, all of these perturbations affected proteins of different sizes in the same proportions, which could thus be described as changes of a well-defined cytoplasmic viscosity. We then observed that the reactivation of ATP-dependent enzymes in diffusionally-impaired cells is sufficient to promote cytoplasmic stirring and diffusion of inert sfGFP. Lastly, we demonstrated that catalytically-induced enhanced diffusion, previously observed only in vitro, can be detected for some ATP-dependent enzymes also in living systems.