Biology subjects

Boersma, A. J.

Publications and source records attributed to Boersma, A. J..

3 recordsLinked to original sources

bioRxiv2023

Genetically-encoded probes to determine nonspecific hydrophobic and electrostatic binding in cells

Proteins interact nonspecifically with other components in the crowded cell through associative interactions. This environmental stickiness alters for example folding stability, protein diffusion, and aggregation propensity. However, the magnitude and variation in nonspecific electrostatic and hydrophobic binding energies in the cell are unclear. Here, we develop genetically-encoded fluorescence excitation ratiometric probes to determine nonspecific binding interactions. We determine hydrophobic and electrostatic interactions by systematically varying a sensing peptide on the probe. The sensors are verified in vitro and tested in HEK293T, where the nonspecific binding is highest for highly cationic and hydrophobic domains. Perturbing the cell by energy depletion increases the dependence of binding strength on peptide electrostatics, showing that the cellular conditions tune the nonspecific interaction architecture in cells. The sensors will allow estimation of nonspecific interactions and how these interactions may change in response to stresses.

biophysics↗

bioRxiv2023

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

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.

biophysics↗

bioRxiv2021

A precise and general FRET-based method for monitoring structural transitions in protein self-organization

Proteins assemble into a tremendous variety of dynamic and functional structures. Sensitive measurements directly in cells with a high spatiotemporal resolution are needed to distinguish these different assemblies. Here, we demonstrate precise and continuous monitoring of cytoplasmic protein self-assemblies and their structural transitions. Intermolecular FRET with both the donor and acceptor protein at the same target protein provides high sensitivity while retaining the advantage of straightforward ratiometric imaging. We measure different assembly structures, transient intermediate states kinetics, and assembly formation resolved in space and time. Specifically, the method recapitulates that i) the mutant Huntingtin exon1 (mHttex1) protein first forms low-FRET and presumably less ordered assemblies in yeast and human cells, which develop into high-FRET aggregates, ii) the chaperone DNAJB6b prevents low-FRET mHttex1 assemblies, yet coassembles with mHttex1 aggregates, and iii) FUS condensates have mutation-dependent nanoscopic structures. FACS measurements allow assembly measurement in a high-throughput manner crucial for screening efforts, while fluorescence microscopy provides spatiotemporally-resolved measurements on the single-condensate level during a cells lifetime to assess the biological consequences. Implementation in other native or non-native proteins could provide insight into many studies involving protein condensation or aggregation.

biophysics↗