Westerlaken, I. | bioRxiv Science

bioRxiv2025

Dps binds and protects DNA in starved Escherichia coli with minimal effect on chromosome accessibility, dynamics and organisation

Dps is the most abundant nucleoid-associated protein in starved Escherichia coli with [~]180,000 copies per cell. Dps binds DNA and oxidises iron, facilitating survival in harsh environments. Dps-DNA complexes can form crystalline structures, leading to the proposed model that Dps reorganises the starved E. coli nucleoid into a compact liquid crystal, slowing chromosome dynamics and limiting access of other proteins to DNA. In this work, we directly tested this model using live-cell super-resolution microscopy and Hi-C analysis. We found that after 96 h of starvation, Dps compacts the nucleoid and increases short-range DNA-DNA interactions, but does not affect chromosome accessibility to large protein nanocages or small restriction enzymes. We also report that chromosome dynamics and organisation are primarily impacted by the bacterial growth phase; the effect of Dps is relatively minor. Our work clarifies the role of Dps in modulating nucleoid properties, and we propose an updated model for Dps-DNA interactions in which Dps binds, protects and compacts DNA largely without influencing chromosome access, dynamics and organisation. Additionally, this work provides a general framework for assessing the impact of nucleoid-associated proteins on key aspects of chromosome function in live cells. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/673347v3_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1385efforg.highwire.dtl.DTLVardef@1e4cb76org.highwire.dtl.DTLVardef@1e9e43dorg.highwire.dtl.DTLVardef@8768f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Full-Length Single-Molecule Protein Fingerprinting

Proteins are the primary functional actors of the cell. Hence, their identification is pivotal to advance our understanding of cell biology and disease. Current protein analysis methods are of limited use for distinguishing proteoforms. In particular, mass spectrometric methods often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate FRET-based single-molecule protein fingerprinting to map the location of individual amino acids and a post-translational modification within single full-length protein molecules. Using an approach that relies on transient binding of fluorescently labeled DNA strands to probe the amino acids on a protein one by one we show that we can fingerprint intrinsically disordered proteins as well as folded globular proteins with sub-nanometer resolution. We anticipate that this technology will be used for proteoform identification in biological and translational research with ultimate sensitivity.

biophysics↗