Genetic information encoded in the DNA sequences is maintained, replicated, and transcribed across the tree of life. Organisms evolved multiple hierarchical layers of chromosome organization which ensure that DNA can be contained but also processed within individual cells. This thesis focuses on SMC (Structural Maintenance of Chromosomes) protein complexes, an evolutionarily conserved family of motor proteins that hold sister chromatids together and fold genomes throughout the cell cycle by DNA loop extrusion. These complexes play a key role in a variety of functions in the packaging and regulation of chromosomes, and they have been intensely studied in recent years. Despite their importance, the detailed molecular mechanism for DNA loop extrusion by SMC complexes remains unresolved. In this thesis, we utilized single-molecule fluorescence and magnetic tweezers to study the intricacies of DNA loop extrusion by SMC complexes in a controlled in vitro environment. We observed, described, and analyzed the behaviour of SMCs which yielded detailed yet important contributions to the resolution of the DNA loop extrusion mechanism as well as to the physiological impact on genome structure. In particular, we observed that large DNA-tethered roadblocks cannot stall loop extrusion efficiently and concluded that DNA loop extrusion can occur in a non-topological manner. In contrast, we found that a small DNA-binding protein, CCCTC-binding factor (CTCF) stalls cohesin through direct protein-protein interactions with cohesin’s N-terminal region. Furthermore, we could reconcile previous results that suggested different loop extrusion mechanisms for different members of the SMC protein family. We demonstrated that all eukaryotic SMCs extrude DNA asymmetrically, i.e. only from one side at a time, and that frequent direction switches yield the previously observed ‘symmetric’ loop extrusion. Furthermore, we showed that DNA supercoiling is intimately linked to loop extrusion for all eukaryotic SMC complexes, postulating a common DNA loop extrusion mechanism.

This doctoral thesis stands on three pillars that emerged from following my scientific interests: i) biophysics, ii) synthetic biology, and iii) biosecurity. The former, biophysics, reflects my desire for deep understanding of biological systems and my affinity for experimental work. The latter, synthetic biology and biosecurity, were born from the conviction that the understanding obtained in pursuing science offers the most fulfillment when applied to the benefits of society.

The main part of this thesis explores methodologies for the extraction, and characterization of large-scale DNA, with a particular focus on the megabase-pair length DNA from bacterial sources. The research aims to bridge the gap between in vivo chromosome studies and in vitro single-molecule techniques by developing approaches that enable the investigation of chromosome structure and dynamics at a more relevant genomic scale. The following chapters detail the experimental approaches, results, and conclusions drawn from this work.

The last few years have shown promising developments in single-molecule protein sequencing using a biological nanopore. Using only one membrane pore in a salt buffer, an applied potential creates a measurable ion current through the pore’s constriction. If a small molecule passes through the pore, this disruption is visible as a characteristic current pattern based on the molecular structure of the molecule. Recent developments by Henry in 2021 show that single amino acid substitution in short proteins can be detected using this method. Unfortunately, due to the complexity of the reading, de novo sequencing does not yet apply to these single molecule reads. Nevertheless, detecting different variants of a protein is possible. In current work this method is being developed to detect phosphorylation sites on a peptide of biological significance. For the latter, an immunopeptide called IRS2 is chosen to be once and twice phosphorylated. This phosphorylation causes the peptide to be a cancer biomarker. If this low-cost method of post-translational modification detection is successful, it would be an improvement compared to the standard mass spectrometry sequencing approach. In this BEP research, problems faced before and after sequencing the IRS2 peptide are approached from two different perspectives. The first aim is to adapt the data acquisition software to automate the workflow. The second aim is to adapt and model the Freely Jointed Chain (FJC) Model to a heterogeneously charged peptide. Both aims are defined to be applicable to other peptides as well. For the first aim, a LabVIEW plugin was developed using insights from data processing in MATLAB. This tool detects the state of the nanopore reading, responds using voltage control in a closed feedback loop and frequently allows for calibration checks. All features were tested with training sequences from real data and show promising results. However, more testing in the lab is required to determine its accuracy compared to a human operator. For the second aim, the FJC model was analytically adapted to the IRS2 peptide inside the nanopore’s electric field. The energetically most favourable configuration was then sought with a Metropolis Algorithm. As a result, the electrostatic potential was calculated and implemented into the Metropolis Algorithm. Despite the simplification of this method, it is still expandable in a modular way to incorporate additional potential such as charge-charge interactions or springs to simulate backbone flexibility. Finally, improving this model further would eventually lead to the relative positions of all chain elements inside the pore to develop an understanding of the (phosphorylated) IRS2 readings.

At first glance nanopores may appear simple, almost intuitive, to understand given that they are, quite literally, ‘just’ very small pores in a membrane. In fact, one may even wonder why at all we need trained scientists to study such seemingly simple entities. The short answer is that nanopores, as the word suggests, are nanoscale entities and, as such, one can not directly see or experience any of the events that occur down there. The long answer can be found in this thesis. Here, I present and discuss a wide array of nanopores, from biological nanopores like the nuclear pore complex (NPC), to solidstate nanopores, and DNA-origami nanopores. While the central focus of my research is to understand the inner workings of the NPC, a short journey into the world of ion transport in solid-state nanopores is first undertaken, with special emphasis on the random fluctuations of the ion flow within the nanopore, referred to as current noise. Next, I introduce the concept of biomimetic nanopores, where a solid-state nanopore is ‘camouflaged’ by coating its inner surface with purified proteins, resulting in an entity that behaves somewhat like a real NPC. Biomimetic nanopores have enabled us to mimic, study, and gain new insights into how the real NPC works, and bear great potential for further developments and discoveries.

In this thesis, we demonstrate optical trapping of HSP90 proteins in plasmonic nanoapertures to study the HSP90 conformational change. This technique is label-free and non-destructive. The resonance of the bow tie shaped plasmonic nanoaperture is used to create a very strong electric field gradient force able to trap proteins. The resonance phenomenon is also used for detecting the trapping events, using the change in transmission that results from the presence of a protein in the trap. HSP90 can be trapped very stably for upwards of 30 seconds. The signal resulting from trapping HSP90 significantly differs from signals from bead traps, especially in the low-frequency regime. We observe strong evidence for a two level system when HSP90 was incubated with AMP-PNP which should slow down the conformational change (fit with R^2=0.9988). More work is required to demonstrate whether this two level system results from the conformational change. We conclude that there is a need for more advanced statistical methods to more conclusively prove that the HSP90 undergoes conformational change between two main levels and suggest further improvements to the experiments.

Cell replication is a fascinating biological process that ensures the proliferation of a species via division of a mother cell into two daughter ones. This process in bacteria is performed by a complex protein machinery named the Z-ring, which assembles at the cell mid-plane and promotes progressive membrane constriction down to division. A central element of this machinery is FtsZ, a protein that polymerizes into filaments that constitute the main scaffold of the ring. Even though this protein was found to be essential, many aspects concerning the Z-ring assembly, the role of FtsZ-associated proteins, and the contribution of FtsZ to the constricting force in the division process are not yet clearly determined. To address these questions, in this thesis we aimed to reconstitute a minimal divisome in vitro, in order to isolate single components from the complex cellular environment, and study their functionality in a bottom-up way. Following this approach, we aimed to better understand the dynamics and the parameters involved in FtsZ filament association into bundles, and to verify whether such structures are capable to generate a force on the lipid membrane.

The function and phenotype of a cell is determined by a complex network of interactions between DNA, RNA, proteins and metabolites. Therefore, a comprehensive approach that integrates genomics, transcriptomics, proteomics, and metabolomics is necessary to achieve full understanding of biological processes and disease. Recent technological developments have mostly focused on the study of genomes. DNA sequencing has become fast, cheap, and ubiquitous. The study of other -omes, especially the proteome, remains expensive and time-consuming...

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 these goals, cells are assisted by proteins that organise the DNA by locally bending the DNA, wrapping DNA around them, or by making DNA loops. A prime example are the Structural Maintenance of Chromosomes (SMC) family of proteins,which is known to be essential for DNA organisation. In eukaryotes, the SMC complex cohesin is responsible for keeping sister-chromatids together until the cell is ready to divide. Without cohesin, division might occur prematurely, leading to unevenly divided DNA. The SMC complex condensin is responsible for compacting the DNA into mitotic chromosomes. Indeed, without condensin, the DNA does not formproperly organised chromosomes. This thesis describes a series of experiments that aim to understand the molecular mechanism of these SMC proteins.

Nuclear pore complexes (NPC’s) facilitate the exchange of macromolecules between the cytoplasm and nucleus and act as a selective barrier for macromolecules in eukaryotes. Ongoing research suggests that the discriminatory function of the NPC is caused by nucleoporins rich in phenylalanine-glycine amino acids residue repeats (FG-regions) which fill the nuclear pore. To determine if the FG-domains are responsible for the selectivity of a NPC, in this research we will mimic a nuclear pore complex by coating a solid state nanopore with yeast FG-nup-NSP1. After coating a wild-type nucleoporin, to a solid-state nanopore, we find that a yeast importer protein kap95 was able to translocate through the nanopore. The dwell times were on the order of tens of milliseconds. An artificial control protein, tCherry, with the same size as kap95, was not able to pass the artificial NPC. By
coating a solid-state nanopore with mutated FG-nup-NSP1, where hydrophobic amino acid residues (F, I, L and V) were replaced with serine, we found that kap95 translocated with much lower translocation times, comparable to an uncoated nanopore, indicating less interaction with the mutated nucleoporins. Furthermore tCherry was also able to pass through the mimicked nuclear pore with mutated nucleoporins, making a very strong argument that FG-repeat regions in nucleoporins are responsible for the selectivity in NPC’s.