Bacterial chromosome organization by ParB proteins

This thesis explores the mechanisms that underlie chromosome organization in bacteria. Bacteria are considered amongst the simplest living organisms on our planet. They lack the cellular organization found in other domains of life (Archaea or Eukaryotics) and often have simpler life cycles. Over the past decade, we gained increasing knowledge...

New Ways to Look through Nanopores

In this thesis, we have introduced palladium zero-mode waveguides (Pd ZMWs) as a valuable tool for biophysics research and have showcased several applications thereof. We started by using them as single-molecule sensors for free translocations of fluorophores, proteins, and voltage-driven translocations of DNA. By studying salt-gradient driven...

Next-Generation Protein Identification: Advancing Single-Molecule Fluorescence Approaches

Proteins are the workhorses of the cell, as such, they form the basis of all living systems. In order to fully understand biological processes, the ability to identify and quantify the proteins in cells is crucial. Identification can be achieved by determining the amino acid sequence of proteins, since this sequence is unique for each protein....

The nucleotide addition cycle of the SARS-CoV-2 polymerase

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We use a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core...

Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective

The absence of 'shovel-ready' anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day,...

CLASP Suppresses Microtubule Catastrophes through a Single TOG Domain

The dynamic instability of microtubules plays a key role in controlling their organization and function, but the cellular mechanisms regulating this process are poorly understood. Here, we show that cytoplasmic linker-associated proteins (CLASPs) suppress transitions from microtubule growth to shortening, termed catastrophes, including those...

Single-molecule approaches to unravel the mechanism of SMC proteins

Every cell deals with the challenge of organising its DNA. First, the DNA needs to be compacted in size by several orders of magnitude. For example, in each human cell, 2 meters of DNA need to fit inside a micron-sized cell nucleus. Second, the DNA needs to stay accessible for cellular processes such as transcription and replication. To achieve...

Single-molecule protein sequencing through fingerprinting: Computational assessment

Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we...

Strand separation establishes a sustained lock at the Tus-Ter replication fork barrier

The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand...