Self-association of a nucleoid-binding protein increases with macromolecular crowding inEscherichia coli

Abstract

Many proteins self-associate to achieve function. Macromolecular crowding enhances protein self-assembly in buffer experiments with added crowders, and crowding could therefore regulate protein function and organization in cells. In eukaryotic cells, protein condensation has been shown to increase with crowding. However, it is unclear what the effect of crowding is on native protein self-assembly in the highly crowded Escherichia coli cell. To determine the role of crowding in the self-assembly of a native protein, we study here the nucleoid-binding H-NS in E. coli and alter macromolecular crowding using a set of perturbations. We followed H-NS self-assembly using a FRET-based method for determining intermolecular interactions with a single genetic intervention. In dilute cell lysate, we see that H-NS self-assembly increases with salts, macromolecular crowding, and its own concentration. In E. coli, the oligomerization increases with crowding. We see that the response of H-NS oligomerization to a sudden crowding change is not immediate but requires time to adapt. Our findings implicate that in-cell crowding affects intracellular organization by promoting self-assembly.

Molecular and thermodynamic determinants of self-assembly and hetero-oligomerization in the enterobacterial thermo-osmo-regulatory protein H-NS

“…DLS experiments on an H-NS ortholog from Salmonella typhimurium report on the possibility of an assembly of 4–8 dimers at 500 μM concentration, with the maximal hydrodynamic radius \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${R}_h$\end{document} ranging between 12–20 nm for 125–500 μM protein concentration range ( 29 ). With the maximal in vivo concentration of H-NS being ∼130 μM, ( 61 ) the octameric assembly is an upper limit under these conditions, though macromolecular crowding enhances oligomerization ( 30 ). We stress here that other oligomeric species exist in equilibrium with the large octameric assembly as can be directly inferred from AUC experiments.…”

“…The molecular features of H-NS that determine functional outcomes are varied and span a range of processes. For example, it has been proposed that at elevated temperatures (say, 310 K, which is the body temperature of a mammalian host), gene repression is eliminated by the disassembly of H-NS oligomers, ( 4 ) while the oligomerization is enhanced by macromolecular crowding ( 30 ) which presumably acts as a protective mechanism under high extracellular osmolarity. H-NS also forms a heteromer with StpA (a paralog) stabilizing it, which is otherwise degraded by the Lon protease ( 15 , 32 ).…”

Macromolecular Crowding, Phase Separation, and Homeostasis in the Orchestration of Bacterial Cellular Functions

“…The lower crowding in mammalian cells has also been confirmed by a macromolecular crowding sensor (unpublished). The biochemical organization can strongly increase macromolecular crowding effects 287 such as increased protein self-assembly. 287 A common source of a change in density and crowding in most cells is osmotic stress.…”

“…The biochemical organization can strongly increase macromolecular crowding effects 287 such as increased protein self-assembly. 287 A common source of a change in density and crowding in most cells is osmotic stress. Fundamentally, the different domains of life have a similar response: release of water to the extracellular environment with a higher osmolality leads to a reduction of cell volume, which increases crowding, ionic strength, and internal osmolality.…”