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Uncontrolled generation of DNA double-strand breaks (DSBs) in cells is regarded as a highly toxic event that threatens cell survival. Radiation-induced DNA DSBs are commonly measured by pulsed-field gel electrophoresis, microscopic evaluation of accumulating DNA damage response proteins (e.g., 53BP1 or gamma-H2AX) or flow cytometric analysis of gamma-H2AX. The advantage of flow cytometric analysis is that DSB formation and repair can be studied in relationship to cell cycle phase or expression of other proteins. However, gamma-H2AX is not able to monitor repair kinetics within the first 60 min postirradiation, a period when most DSBs undergo repair. A key protein in non-homologous end joining repair is the catalytic subunit of DNA-dependent protein kinase. Among several phosphorylation sites of DNA-dependent protein kinase, the threonine at position 2609 (T2609), which is phosphorylated by ataxia telangiectasia mutated (ATM) or DNA-dependent protein kinase catalytic subunit itself, activates the end processing of DSB. Using flow cytometry, we show here that phosphorylation at T2609 is faster in response to DSBs than gamma-H2AX. Furthermore, flow cytometric analysis of T2609 resulted in a better representation of fast repair kinetics than analysis of gamma-H2AX. In cells with reduced ligase IV activity, and wild-type cells where DNA-dependent protein kinase activity was inhibited, the reduced DSB repair capacity was observed by T2609 evaluation using flow cytometry. In conclusion, flow cytometric evaluation of DNA-dependent protein kinase T2609 can be used as a marker for early DSB repair and gives a better representation of early repair events than analysis of gamma-H2AX.

Time-resolved cryogenic electron microscopy (cryo-EM) is a powerful technique for capturingtransient molecular and viral structures during conformational changes. This method providesunique and potentially critical insights into the transient states of biomolecules, which can beinvaluable for drug development. Additionally, it offers glimpses into the pathology of viruses asthey interact with their immediate environment. In this project, a plunge freezer initially designedfor a spray-and-plunge approach in time-resolved cryo-EM [24] was modified to implement aflash-and-freeze system [3]. This modified system was tested on two different viruses: influenzaH3N2 type A and Chaetoceros tenuissimus DNA virus type II. The primary objective was tovisualize an intermediate state during the membrane fusion process between influenza A andliposomes with endosomal characteristics. Although no intermediate state was captured forinfluenza, the activation and pH reduction were successfully achieved. The study on Chaetocerostenuissimus DNA virus type II focused on potential conformational changes due to a drop in pH,rather than capturing intermediate states in a time-resolved manner. Future experiments withmore precise control over blotting, delivered intensity, and the concentration of the cagedcompound are expected to facilitate the capture and analysis of intermediate states.

Measuring colocalisation is not straightforward with a plethora of coefficients that encapsulate different definitions. Measurements may also be implemented differently. Not only do measurements differ; interconversion is impossible making comparisons challenging. There is a need to cull coefficients and for clear definitions of what precisely is meant by colocalisation in individual studies. Colocalisation can be considered to have two components; co-occurrence which reports whether the fluorophores are found together and correlation which reports on the similarity in their patterns of intensity.

Nanofluidic channels have become a versatile tool to manipulate single DNA molecules. They allow investigation of confined single DNA molecules from a fundamental polymer physics perspective as well as for example in DNA barcoding techniques.

The advent of machine learning-based structure prediction algorithms such as AlphaFold2 (AF2) and RoseTTa Fold have moved the generation of accurate structural models for the entire cellular protein machinery into the reach of the scientific community. However, structure predictions of protein complexes are based on user-provided input and may require experimental validation. Mass spectrometry (MS) is a versatile, time-effective tool that provides information on post-translational modifications, ligand interactions, conformational changes, and higher-order oligomerization. Using three protein systems, we show that native MS experiments can uncover structural features of ligand interactions, homology models, and point mutations that are undetectable by AF2 alone. We conclude that machine learning can be complemented with MS to yield more accurate structural models on a small and large scale.

DNA intercalators that have high affinity and slow kinetics are developed for potential DNA-targeted therapeutics. Although many natural intercalators contain multiple chiral subunits, only intercalators with a single chiral unit have been quantitatively probed. Dumbbell-shaped DNA threading intercalators represent the next order of structural complexity relative to simple intercalators, and can provide significant insights into the stereoselectivity of DNA-ligand intercalation. We investigated DNA threading intercalation by binuclear ruthenium complex [mu-dppzip(phen)(4)Ru-2](4+) (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)(2)dppz) and the distal subunit (Ru(phen)(2)ip), respectively, each of which can be either right-handed (Delta) or left-handed (Lambda). We used optical tweezers to measure single DNA molecule elongation due to threading intercalation, revealing force-dependent DNA intercalation rates and equilibrium dissociation constants. The force spectroscopy analysis provided the zero-force DNA binding affinity, the equilibrium DNA-ligand elongation Delta x(eq), and the dynamic DNA structural deformations during ligand association x(on) and dissociation x(off). We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a K-d of 27 +/- 3 nM for (Delta,Lambda)-Piz to a K-d of 622 +/- 55 nM for (Lambda,Delta)-Piz. The striking affinity decrease is correlated with increasing Delta x(eq) from 0.30 +/- 0.02 to 0.48 +/- 0.02 nm and x(on) from 0.25 +/- 0.01 to 0.46 +/- 0.02 nm, but limited x(off) changes. Notably, the affinity and threading kinetics is 10-fold enhanced for right-handed intercalating subunits, and 2- to 5-fold enhanced for left-handed distal subunits. These findings demonstrate sterically dispersed transition pathways and robust DNA structural recognition of chiral intercalators, which are critical for optimizing DNA binding affinity and kinetics.

The aim of this study was to determine the thickness of the hydrodynamic diffusion layer (h(HDL)) of three poor water-soluble compounds under laminar fluid flow using a single particle dissolution technique. The single particle dissolution experiments were performed in a flowing aqueous medium using four different fluid velocities (v), ranging from 46 to 103 mm/s. The particles used had an initial radius (r) of 18.8 to 52.3 mu m. The determined h(HDL) values were calculated from both dissolution experiments and computational fluid dynamics (CFD) simulation. In this study, single particle dissolution experiments gave, with one exception, h(HDL) values in the range of 2.09 to 8.85 mu m and corresponding simulations gave h(HDL) values in the range of 2.53 to 4.38 mu m. Hence, we found a semi-quantitative concordance between experimental and simulated determined h(HDL) values. Also, a theoretical relation between the dependence of hHDL on particle radius and flow velocity of the medium was established by a series of CFD simulations in a fluid velocity range of 10-100 mm/s and particle size (radius) range of 5-40 mu m. The outcome suggests a power law relation of the form h(HDL)alpha r(3/5)v(-2/5). In addition, the h(HDL) seems to be independent of the solubility, while it has a diffusion coefficient dependence. In conclusion, the hHDL values were determined under well-defined conditions; hence, this approach can be used to estimate the h(HDL) under different conditions to increase the understanding of the mass transfer mechanisms during the dissolution process.

We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.

The worldwide obesity epidemic(1) makes it important to understand how lipid turnover (the capacity to store and remove lipids) regulates adipose tissue mass. Cross-sectional studies have shown that excess body fat is associated with decreased adipose lipid removal rates(2,3). Whether lipid turnover is constant over the life span or changes during long-term weight increase or loss is unknown. We determined the turnover of fat cell lipids in adults followed for up to 16 years, by measuring the incorporation of nuclear bomb test-derived C-14 in adipose tissue triglycerides. Lipid removal rate decreases during aging, with a failure to reciprocally adjust the rate of lipid uptake resulting in weight gain. Substantial weight loss is not driven by changes in lipid removal but by the rate of lipid uptake in adipose tissue. Furthermore, individuals with a low baseline lipid removal rate are more likely to remain weight-stable after weight loss. Therefore, lipid turnover adaptation might be important for maintaining pronounced weight loss. Together these findings identify adipose lipid turnover as an important factor for the long-term development of overweight/obesity and weight loss maintenance in humans.

Chemotherapy drugs (CDs) disrupt the lipid membrane's insulation properties by inducing stable ion pores across bilayer membranes. The underlying molecular mechanisms behind pore formation have been revealed in this study using several methods that confirm molecular interactions and detect associated energetics of drugs on the cell surface in general and in lipid bilayers in particular. Liposome adsorption and cell surface binding of CD colchicine has been demonstrated experimentally. Buffer dissolved CDs were considerably adsorbed in the incubated phospholipid liposomes, measured using the patented 'direct detection method'. The drug adsorption process is regulated by the membrane environment, demonstrated in cholesterol-containing liposomes. We then detailed the phenomenology and energetics of the low nanoscale dimension cell surface (membrane) drug distribution, using atomic force microscopy (AFM) imaging what addresses the surface morphology and measures adhesion force (reducible to adhesive energy). Liposome adsorption and cell surface binding data helped model the cell surface drug distribution. The underlying molecular interactions behind surface binding energetics of drugs have been addressed in silico numerical computations (NCs) utilizing the screened Coulomb interactions among charges in a drug-drug/lipid cluster. Molecular dynamics (MD) simulations of the CD-lipid complexes detected primarily important CD-lipid electrostatic and van der Waals (vdW) interaction energies. From the energetics point of view, both liposome and cell surface membrane adsorption of drugs are therefore obvious findings. Colchicine treated cell surface AFM images provide a few important phenomenological conclusions, such as drugs bind generally with the cell surface, bind independently as well as in clusters of various sizes in random cell surface locations. The related adhesion energy decreases with increasing drug cluster size before saturating for larger clusters. MD simulation detected electrostatic and vdW and NC-derived charge-based interactions explain molecularly of the cause of cell surface binding of drugs. The membrane binding/association of drugs may help create drug-lipid complexes with specific energetics and statistically lead to the creation of ion channels. We reveal here crucial molecular understanding and features of the pore formation inside lipid membranes that may be applied universally for most of the pore-forming existing agents and novel candidate drugs.

The aim of this study was to investigate the relationship between ordered plasma membrane nanodomains and actin filaments using di-4-ANEPPDHQ and laurdan together with the reagents that affect actin filament dynamics in live Jurkat and primary T cells. The degree of lipid packing can be quantified using polarity sensitive membrane dyes such as laurdan and di-4-ANEPPDHQ. These two dyes display a red shift in their emission peaks for membranes in ld phase relative to lo phase. Laurdan is uncharged and can easily flip between two leaflets of the plasma membrane and we demonstrate that it reports equally on the two leaflets of the plasma membrane.

Membrane nanodomains are dynamic liquid entities surrounded by another type of dynamic liquid. Diffusion can take place inside, around and in and out of the domains, and membrane components therefore continuously shift between domains and their surroundings. In the plasma membrane, there is the further complexity of links between membrane lipids and proteins both to the extracellular matrix and to intracellular proteins such as actin filaments. In addition, new membrane components are continuously delivered and old ones removed. On top of this, cells move. Taking all of this into account imposes great methodological challenges, and in the present chapter we discuss some methods that are currently used for membrane nanodomain studies, what information they can provide and their weaknesses.

Viruses are entities which are made of a few genes and are reliant on obligate parasitism to propagate. Due to the obligate connection to their hosts, virus evolution is constrained to the type of host. Viruses however do transmit to evolutionary distinct hosts; in these cases, the phylogenetic relationship of the hosts usually are close. In some instances, RNA-viruses have made host jumps between evolutionary distant hosts, such as the host jump from invertebrates to vertebrates, and fungi to arthropod. Partitiviruses are double stranded RNA viruses which mainly infect fungi and plants. The defining characteristic of these double stranded RNA viruses are the double layered capsids which are formed by a single open reading frame (ORF). The capsid proteins form icosahedral virus particles which are in the magnitude of 30-40 nm. Metagenomic studies have discovered partitiviruses originating from an insect in the Odanata family, a finding which contradicts the fungal host specificity of partitiviruses. The finding of the Hubei.PLV 11 thus implies the existence of a partitiviruses containing structural elements in their capsids which could be involved in the infection of arthropods. Thus, this virus could be used as a model for a structural comparison with its fungi infecting relatives with hopes to identify common viral structural factors necessary for the infection of arthropods. For this purpose, the Hubei.PLV ORF was cloned and then transfected into insect Spodoptera frugiperda (Sf-9) cells using a baculovirus expression system, “bac-to-bac” expression system. The FLAG-tagged capsid proteins were expressed by the Sf-9 cells to be approximately 60 kDa. After ultra-centrifugation in a sucrose gradient, some spontaneous assembly into the expected ~40 nm icosahedral virus-like particles were observed using low resolution scanning electron microscopy. The observed particles were also confirmed by a dynamic light scattering experiment (DLS) and a higher resolution cryo-EM microscope. Thus, the bac-to-bac expression system can be used to produce VLPs from this genus of viruses, and this metagenomically derived virus genome. However, for future success in defining a high-resolution model of this virus, it is recommended that the Sf-9 culture volume is sufficiently high for enough particle production which is necessary for a high-resolution map. The other virus, the Southern pygmy squid Flavilike virus (SpSFV) has been suggested to be the oldest relative of the land based flaviviruses. The SpSFV was found to be the most divergent of the flaviviruses, and to infect invertebrates. Solving for the structure of the SpSFV and comparing it to vertebrate infecting flaviviruses could therefore lead to the identification of factors necessary for the adaptation to vertebrates and thus the humoral immunity by flaviviruses. The soluble E-protein was expressed using the bac-to-bac expression system. The protein was indicated to be multiglycosylated and approximately 50 kDa which is in line with other strains in the genus. Affinity chromatography did not elute this protein, likely due to the His-tag not being spatially available. Cation exchange could elute some protein, but not much from the small ~30 mL culture. To conclude, VLP assembly was confirmed by the Hubei.PLV, thus, solving for the structure is a distinct possibility when a larger Sf-9 culture is used to produce the VLPs. For the SpSFV soluble E-protein, the protein is secreted into the supernatant of the Sf-9 cultures, making purification a possibility. For this, a large Sf-9 culture can be used to produce this protein and then purify it with a cat-ion exchange chromatography.

The protection of the viral genome during extracellular transport is an absolute requirement for virus survival and replication. In addition to the almost universal proteinaceous capsids, certain viruses add a membrane layer that encloses their double-stranded (ds) DNA genome within the protein shell. Using the membrane-containing enterobacterial virus PRD1 as a prototype, and a combination of nanoindentation assays by atomic force microscopy and finite element modelling, we show that PRD1 provides a greater stability against mechanical stress than that achieved by the majority of dsDNA icosahedral viruses that lack a membrane. We propose that the combination of a stiff and brittle proteinaceous shell coupled with a soft and compliant membrane vesicle yields a tough composite nanomaterial well-suited to protect the viral DNA during extracellular transport.

Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth's climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean.

We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our experiments, 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresolutionmicroscopy, MINFLUXattained similar to 1-nanometer precision, resolving molecules only 6 nanometers apart. MINFLUX tracking of single fluorescent proteins increased the temporal resolution and the number of localizations per trace by a factor of 100, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli. As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.

RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.

Over the past 40 years, it has been demonstrated that many enzymes are capable of promiscuous catalytic activities, facilitating the turnover of more than one chemically distinct substrate. This has been argued to play an important role in enzyme evolution, with highly promiscuous progenitor enzymes evolving under evolutionary pressure to modern day specialists, while still retaining some level of their former promiscuous activities¹. This theory has been extensively tested by different experiments using in vitro evolution². The alkaline phosphatase superfamily members provide a particularly attractive showcase for studying enzyme promiscuity, as they often show reciprocal promiscuity, in that the native reaction for one member is often a side-reaction for another³. While deceptively similar, their catalyzed reactions (cleavage of P-O and S-O bonds) proceed via distinct transition states and protonation requirements^4,5. We present detailed computational studies of the promiscuous catalytic activity of three evolutionarily related members: the arylsulfatase from Pseudomonas aeruginosa⁶, and the phosphonate monoester hydrolases from Burkholderia caryophili⁷and Rhizobium leguminosarum⁸. By tracking their structural and electrostatic features, and comparing to other known members of the superfamily, we provide an atomic-level map for functional evolution within this superfamily.

Atomistic simulations of three different proteins at different concentrations are performed to obtain insight into protein mobility as a function of protein concentration. We report on simulations of proteins from diluted to the physiological water concentration (about 70% of the mass). First, the viscosity was computed and found to increase by a factor of 7-9 going from pure water to the highest protein concentration, in excellent agreement with in vivo nuclear magnetic resonance results. At a physiological concentration of proteins, the translational diffusion is found to be slowed down to about 30% of the in vitro values. The slow-down of diffusion found here using atomistic models is slightly more than that of a hard sphere model that neglects the electrostatic interactions. Interestingly, rotational diffusion of proteins is slowed down somewhat more (by about 80-95% compared to in vitro values) than translational diffusion, in line with experimental findings and consistent with the increased viscosity. The finding that rotation is retarded more than translation is attributed to solvent-separated clustering. No direct interactions between the proteins are found, and the clustering can likely be attributed to dispersion interactions that are stronger between proteins than between protein and water. Based on these simulations, we can also conclude that the internal dynamics of the proteins in our study are affected only marginally under crowding conditions, and the proteins become somewhat more stable at higher concentrations. Simulations were performed using a force field that was tuned for dealing with crowding conditions by strengthening the protein-water interactions. This force field seems to lead to a reproducible partial unfolding of an alpha-helix in one of the proteins, an effect that was not observed in the unmodified force field.

In the context of studies of proteins under crowding conditions, it was found that there is a tendency of simulated proteins to coagulate in a seemingly unphysical manner. This points to an imbalance in the protein-protein or protein-water interactions. One way to resolve this is to strengthen the protein-water Lennard-Jones interactions. However, it has also been suggested that dispersion interactions may have been systematically overestimated in force fields due to parameterization with a short cutoff. Here, we test this proposition by performing simulations of liquids and of proteins in solution with systematically reduced C-6 (dispersion constant in a 12-6 Lennard-Jones potential) and evaluate the properties. We find that simulations of liquids with either a dispersion correction or explicit long-range Lennard-Jones interactions need little or no correction to the dispersion constant to reproduce the experimental density. For simulations of proteins, a significant reduction in the dispersion constant is needed to reduce the coagulation, however. Because the protein- and liquid force fields share atom types, at least to some extent, another solution for the coagulation problem may be needed, either through including explicit polarization or through strengthening protein-water interactions.

In recent years, advances in Artificial Intelligence and experimental techniques have revolutionized the field of structural biology. X-ray crystallography and Cryo-EM have provided unprecedented insights into the structures of biomolecules, while the unexpected success of AlphaFold has opened up new avenues of investigation. However, studying the dynamics of proteins at high resolution remains a significant obstacle, especially for fast dynamics. Single-particle imaging (SPI) or Flash X-ray Imaging (FXI) is an emerging technique that may enable the mapping of the conformational landscape of biological molecules at high resolution and fast time scale. This thesis discusses the potential of SPI/FXI, its challenges, recent experimental successes, and the advancements driving its development. In particular, machine learning and neural networks could play a vital role in fostering data analysis and improving SPI/FXI data processing. In Paper I, we discuss the problem of noise and detector masks in collecting FXI data. I simulated a dataset of diffraction patterns and used it to train a Convolutional Neural Network (U-Net) to restore data by denoising and filling in detector masks. As a natural continuation of this work, I trained another machine learning model in Paper II to estimate 2D protein densities from diffraction intensities. In the final chapter, corresponding to Paper III, we discuss another experimental method, time-resolved Small Angle X-ray Scattering (SAXS), and a new algorithm recently developed for SAXS data, the DENsity from Solution Scattering (DENSS) algorithm. I discuss the potential of DENSS in time-resolved SAXS and its application for structural fitting of AsLOV2, a Light-Oxygen-Voltage (LOV) protein domain from Avena sativa.

The helical structure of DNA imposes constraints on the rate of diffusion-limited protein binding. Here we solve the reaction-diffusion equations for DNA-like geometries and extend with simulations when necessary. We find that the helical structure can make binding to the DNA more than twice as fast compared to a case where DNA would be reactive only along one side. We also find that this rate advantage remains when the contributions from steric constraints and rotational diffusion of the DNA-binding protein are included. Furthermore, we find that the association rate is insensitive to changes in the steric constraints on the DNA in the helix geometry, while it is much more dependent on the steric constraints on the DNA-binding protein. We conclude that the helical structure of DNA facilitates the nonspecific binding of transcription factors and structural DNA-binding proteins in general.

Radiation damage limits the resolution in imaging experiments. Damage is caused by energy deposited into the sample during exposure. Ultrashort and extremely bright X-ray pulses from free-electron lasers (FELs) offer the possibility to outrun key damage processes, and temporarily improve radiation tolerance. Theoretical models indicate that high detail-resolutions could be realized on non-crystalline samples with very short pulses, before plasma expansion.

Studies presented here describe the interaction of a very intense and ultrashort X-ray pulse with material, and investigate boundary conditions for flash diffractive imaging both theoretically and experimentally. In the hard X-ray regime, predictions are based on particle simulations with a continuum formulation that accounts for screening from free electrons.

First experimental results from the first soft X-ray free-electron laser, the FLASH facility in Hamburg, confirm the principle of flash imaging, and provide the first validation of our theoretical models. Specifically, experiments on nano-fabricated test objects show that an interpretable image can be obtained to high resolution before the sample is vaporized. Radiation intensity in these experiments reached 10^14 W/cm^2, and the temperature of the sample rose to 60000 Kelvin after the 25 femtosecond pulse left the sample. Further experiments with time-delay X-ray holography follow the explosion dynamics over some picoseconds after illumination.

Finally, this thesis presents results from biological flash-imaging studies on living cells. The model is based on plasma calculations and fluid-like motions of the sample, supported by the time-delay measurements. This study provides an estimate for the achievable resolutions as function of wavelength and pulse length. The technique was demonstrated by our team in an experiment where living cells were exposed to a single shot from the FLASH soft X-ray laser.

Alzheimer’s disease (AD) is a devastating neurodegenerative disease that affect millions of people worldwide. Aggregation of Amyloid-β (Aβ) monomers create toxic oligomers that can interact with cellular membranes and disturb cellular functions, resulting in cell death and neurological dysfunction. Increased levels of oxidative stress have been shown in the brains of AD patients, something that besides the obvious cell and tissue toxicity, also favors the amyloidogenic pathway and generates more Aβ monomers. Here we show that Edaravone, a free radical scavenger can affect the aggregation rate of different lengths of Aβ. We show that Aβ-40 that is more commonly found in vivo aggregates faster with addition of Edaravone, while Aβ-42 aggregates slower or not at all. These findings add up to previous findings where free radical scavengers and antioxidants such as Edaravone have been suggested as a potential treatment in Alzheimer’s disease.

Green fluorescent proteins (GFPs) are ubiquitous for protein tagging and live-cell imaging. Split-GFPs are widely used to study protein-protein interactions by fusing proteins of interest to split GFP fragments that create a fluorophore upon typically irreversible complementation. Thus, controlled dissociation of the fragments is desirable. Although we have found that split strands can be photodissociated, the quantum efficiency of light-induced photodissociation of split GFPs is low. Traditional protein engineering approaches to increase efficiency, including extensive mutagenesis and screening, have proved difficult to implement. To reduce the search space, key states in the dissociation process are modeled by combining classical and enhanced sampling molecular dynamics with QM/MM calculations, enabling the rational design and engineering of split GFPs with up to 20-fold faster photodissociation rates using non-intuitive amino acid changes. This demonstrates the feasibility of modeling complex molecular processes using state-of-the-art computational methods, and the potential of integrating computational methods to increase the success rate in protein engineering projects.

The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PIA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. (C) 2013 Elsevier B.V. All rights reserved.

Biomolecular crowding affects the biophysical and biochemical behavior of macromolecules compared with the dilute environment in experiments on isolated proteins. Computational modeling and simulation are useful tools to study how crowding affects the structural dynamics and biological properties of macromolecules. With increases in computational power, modeling and simulation of large-scale all-atom explicit-solvent models of the prokaryote cytoplasm have now become possible. In this work, we built an atomistic model of the cytoplasm of Escherichia coli composed of 1.5 million atoms and submitted it to a total of 3 mu s of molecular dynamics simulations. The model consisted of eight different proteins representing about 50% of the cytoplasmic proteins and one type of t-RNA molecule. Properties of biomolecules under crowding conditions were compared with those from simulations of the individual compounds under dilute conditions. The simulation model was found to be consistent with experimental data about the diffusion coefficient and stability of macromolecules under crowded conditions. In order to stimulate further work, we provide a Python script and a set of files to enable other researchers to build their own E. coli cytoplasm models to address questions related to crowding.

The rise of antibiotic-resistant bacterial infections poses a global threat. Antibiotic resistance development is generally studied in batch cultures which conceals the heterogeneity in cellular responses. Using single-cell imaging, we studied the growth response of Escherichia coli to sub-inhibitory and inhibitory concentrations of nine antibiotics. We found that the heterogeneity in growth increases more than what is expected from growth rate reduction for three out of the nine antibiotics tested. For two antibiotics (rifampicin and nitrofurantoin), we found that sub-populations were able to maintain growth at lethal antibiotic concentrations for up to 10 generations. This perseverance of growth increased the population size and led to an up to 40-fold increase in the frequency of antibiotic resistance mutations in gram-negative and gram-positive species. We conclude that antibiotic perseverance is a common phenomenon that has the potential to impact antibiotic resistance development across pathogenic bacteria.

The oxidative stress-related reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) have been shown to promote formation of α-synuclein oligomers in vitro. However, the changes in secondary structure of α-synuclein and the kinetics of the oligomerization process are not known and were the focus of this study. Size exclusion chromatography showed that after 1 h of incubation, HNE induced the formation of an oligomeric α-synuclein peak with a molecular weight of about ∼2000 kDa, which coincided with a decreasing ∼50 kDa monomeric peak. With prolonged incubation (up to 24 h) the oligomeric peak became the dominating molecular species. In contrast, in the presence of ONE, a ∼2000 oligomeric peak was exclusively observed after 15 min of incubation and this peak remained constant with prolonged incubation. Western blot analysis of HNE-induced α-synuclein oligomers showed the presence of monomers (15 kDa), SDS-resistant low molecular (30-160 kDa) and high molecular weight oligomers (≥260 kDa), indicating that the oligomers consisted of both covalent and non-covalent protein. In contrast, ONE-induced α-synuclein oligomers only migrated as covalent cross-linked high molecular-weight material (≥300 kDa). Both circular dichroism (CD) and Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy showed that the formation of HNE- and ONE-induced oligomers coincided with a spectral change from random coil to β-sheet. However, ONE-induced α-synuclein oligomers exhibited a slightly higher degree of β-sheet. Taken together, our results indicate that both HNE and ONE induce a change from random coil to β-sheet structure that coincides with the formation of α-synuclein oligomers. Albeit through different kinetic pathways depending on the degree of cross-linking.

Radiation damage is an unavoidable process when performing structural investigations of biological macromolecules with X-rays. In crystallography this process can be limited through damage distribution in a crystal, while for single molecular imaging it can be outrun by employing short intense pulses. Secondary electron generation is crucial during damage formation and we present a study of urea, as model for biomaterial. From first principles we calculate the band structure and energy loss function, and subsequently the inelastic electron cross-section in urea. Using Molecular Dynamics simulations, we quantify the damage and study the magnitude and spatial extent of the electron cloud coming from an incident electron, as well as the dependence with initial energy.

Identifying the protein content in a cell in a fast and reliable manner has become a relevant goal in the field of proteomics. This thesis computationally explores the potential for silicon nitride nanopores to sense and distinguish single miniproteins, which are small domains that promise to facilitate the systematic study of larger proteins. Sensing and identification of these biomolecules using nanopores happens by studying modulations in ionic current during translocation. The approach taken in this work was to study two miniproteins of similar geometry, using a cylindrical-shaped pore. I employed molecular mechanics to compare occupied pore currents computed based on the trajectory of ions. I further used density functional theory along with relative surface accessibility values to compute changes in interaction energies for single amino acids and obtain relative dwell times. While the protein remained inside the nanopore, I found no noticeable differences in the occupied pore currents of the two miniproteins for systems subject to 0.5 and 1.0 V bias voltages. Dwell times were estimated based on the translocation time of a protein that exhibits no interaction with the pore walls. I found that both miniproteins feel an attractive force to the pore wall and estimated their relative dwell times to differ by one order of magnitude. This means even in cases where two miniproteins are indistinguishable by magnitude changes in the ionic current, the dwell time might still be used to identify them. This work was an initial investigation that can be further developed to increase the accuracy of the results and be expanded to assess other miniproteins with the goal to aid future experimental work.

The four-electron reduction of dioxygen to water is the most exothermic non-photochemical reaction available to biology. A detailed molecular description of this reaction is needed to understand oxygen-based redox processes. Horseradish peroxidase (HRP) is a haem-containing redox enzyme capable of catalysing the reduction of dioxygen to water. We developed instrumentation and experimental methodology to capture and characterise by X-ray crystallography transient reaction intermediates in this reaction.

An instrument was designed (“the vapour stream system”) to facilitate reaction initiation, monitoring and intermediate trapping. In combination with single crystal microspectrophotometry, it was used to obtain conditions for capturing a reactive dioxygen complex in HRP. X-ray studies on oxidised intermediates can be difficult for various reasons. Electrons re-distributed in the sample through the photoelectric effect during X-ray exposure can react with high-valency intermediates. In order to control such side reactions during data collection, we developed a new method based on an angle-resolved spreading of the X-ray dose over many identical crystals. Composite data sets built up from small chunks of data represent crystal structures which received different X-ray doses. As the number of electrons liberated in the crystal is dose dependent, this method allows us to observe and drive redox reactions electron-by-electron in the crystal, using X-rays.

The methods developed here were used to obtain a three-dimensional movie on the X-ray-driven reduction of dioxygen to water in HRP. Separate experiments established high resolution crystal structures for all intermediates, showing such structures with confirmed redox states for the first time.

Activity of HRP is influenced by small molecule ligands, and we also determined the structures of HRP in complex with formate, acetate and carbon monoxide.

Other studies established conditions for successfully trapping the M-intermediate in crystals of mutant bacteriorhodopsin, but the poor diffraction quality of these crystals prevented high-resolution structural studies.

The in vitro biological characterization of biomaterials is largely based on static cell cultures. However, for highly reactive biomaterials such as calcium-deficient hydroxyapatite (CDHA), this static environment has limitations. Drastic alterations in the ionic composition of the cell culture medium can negatively affect cell behavior, which can lead to misleading results or data that is difficult to interpret. This challenge could be addressed by a microfluidics-based approach (i.e. on-chip), which offers the opportunity to provide a continuous flow of cell culture medium and a potentially more physiologically relevant microenvironment. The aim of this work was to explore microfluidic technology for its potential to characterize CDHA, particularly in the context of inflammation. Two different CDHA substrates (chemically identical, but varying in microstructure) were integrated on-chip and subsequently evaluated. We demonstrated that the on-chip environment can avoid drastic ionic alterations and increase protein sorption, which was reflected in cell studies with RAW 264.7 macrophages. The cells grown on-chip showed a high cell viability and enhanced proliferation compared to cells maintained under static conditions. Whereas no clear differences in the secretion of tumor necrosis factor alpha (TNF-α) were found, variations in cell morphology suggested a more anti-inflammatory environment on-chip. In the second part of this study, the CDHA substrates were loaded with the drug Trolox. We showed that it is possible to characterize drug release on-chip and moreover demonstrated that Trolox affects the TNF-α secretion and morphology of RAW 264.7 ​cells. Overall, these results highlight the potential of microfluidics to evaluate (bioactive) biomaterials, both in pristine form and when drug-loaded. This is of particular interest for the latter case, as it allows the biological characterization and assessment of drug release to take place under the same dynamic in vitro environment.

Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from human embryonic kidney 293, cord blood mesenchymal stromal and human acute monocytic leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63, and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having average diameters of <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.

The work presented in this thesis describes how structural information about a protein can be acquired, and how it can be used to answer scientific questions about proteins’ function, their dynamic behaviour and their interactions with other proteins or ligands.

The catalytic function of the pyrimidine-degrading, drug metabolizing enzyme β-ureidopropionase (βUP) is dependent on the shift between oligomeric states. Substitution of amino acids H173 and H307 in the dimer-dimer interface and E207Q in the active site revealed that these are crucial for βUP activation. Inhibition studies of substrate-and product analogues allowed for a hypothesis that the ability to interact with F205 might distinguish activators from inhibitors. The first structure of the activated higher oligomer state of human βUP was determined using cryogenic electron microscopy, and confirmed that the closed entrance loop conformations and dimer-dimer interfaces are conserved between HsβUP and DmβUP. 

Interactions between the epigenetic drug target SET and MYND domain containing protein 3 (SMYD3) and possible inhibitors were investigated. A crystal structure confirmed the covalent bond of a rationally designed, targeted inhibitor to C186 in the active site of SMYD3. A new allosteric binding site was discovered using a biosensor screen with a blocked active site. Crystal structures revealed the location of the new binding site, and the binding mode of the (S)-and (R) enantiomers of the allosteric inhibitor. Lastly, a fragment based drug discovery approach was taken, co-crystallizing and soaking SMYD3 with hits from a fragment screen. This resulted in four crystal structures with weak electron density of fragments at several locations in the enzyme. 

The dynamic acetylcholine binding protein (AChBP) is a homologue of a Cys-loop type ligand gated ion channel. Hits from various biosensor screens, of which some indicated conformational changes, were co-crystallized with AChBP. Seven crystal structures of AChBP in complex with hit compounds from the biophysical screens were determined. Small conformational changes in the Cys-loop were detected in several of the crystal structures, coinciding with the results from the biosensor screens.

In these studies, we explore new strategies for the investigation of the function and regulation of proteins relevant in drug discovery and optimization.

X-ray free-electron lasers have opened up the possibility of structure determination of protein crystals at room temperature, free of radiation damage. The femtosecond-duration pulses of these sources enable diffraction signals to be collected from samples at doses of 1000 MGy or higher. The sample is vaporized by the intense pulse, but not before the scattering that gives rise to the diffraction pattern takes place. Consequently, only a single flash diffraction pattern can be recorded from a crystal, giving rise to the method of serial crystallography where tens of thousands of patterns are collected from individual crystals that flow across the beam and the patterns are indexed and aggregated into a set of structure factors. The high-dose tolerance and the many-crystal averaging approach allow data to be collected from much smaller crystals than have been examined at synchrotron radiation facilities, even from radiation-sensitive samples. Here, we review the interaction of intense femtosecond X-ray pulses with materials and discuss the implications for structure determination. We identify various dose regimes and conclude that the strongest achievable signals for a given sample are attained at the highest possible dose rates, from highest possible pulse intensities.

Knowledge of the manganese oxidation states of the oxygenevolving Mn4CaO5 cluster in photosystem II (PSII) is crucial toward understanding the mechanism of biological water oxidation. There is a 4 decade long debate on this topic that historically originates from the observation of a multiline electron paramagnetic resonance (EPR) signal with effective total spin of S = 1/2 in the singly oxidized S-2 state of this cluster. This signal implies an overall oxidation state of either Mn(III)(3)Mn(IV) or Mn(III)Mn(IV)(3) for the S-2 state. These 2 competing assignments are commonly known as "low oxidation (LO)" and "high oxidation (HO)" models of the Mn4CaO5 cluster. Recent advanced EPR and Mn K-edge X-ray spectroscopy studies converge upon the HO model. However, doubts about these assignments have been voiced, fueled especially by studies counting the number of flash-driven electron removals required for the assembly of an active Mn4CaO5 cluster starting from Mn(II) and Mnfree PSII. This process, known as photoactivation, appeared to support the LO model since the first oxygen is reported to evolve already after 7 flashes. In this study, we improved the quantum yield and sensitivity of the photoactivation experiment by employing PSII microcrystals that retained all protein subunits after complete manganese removal and by oxygen detection via a custom built thin-layer cell connected to a membrane inlet mass spectrometer. We demonstrate that 9 flashes by a nanosecond laser are required for the production of the first oxygen, which proves that the HO model provides the correct description of the Mn4CaO5 cluster's oxidation states.

Kinetochores are protein complexes on the chromosomes, whose function as linkers between spindle microtubules and chromosomes is crucial for proper cell division. The mechanisms that facilitate kinetochore capture by microtubules are still unclear. In the present study, we combine experiments and theory to explore the mechanisms of kinetochore capture at the onset of meiosis I in fission yeast. We show that kinetochores on homologous chromosomes move together, microtubules are dynamic and pivot around the spindle pole, and the average capture time is 3-4 minutes. Our theory describes paired kinetochores on homologous chromosomes as a single object, as well as angular movement of microtubules and their dynamics. For the experimentally measured parameters, the model reproduces the measured capture kinetics and shows that the paired configuration of kinetochores accelerates capture, whereas microtubule pivoting and dynamics have a smaller contribution. Kinetochore pairing may be a general feature that increases capture efficiency in meiotic cells.

The biological influence of radiation on living matter has been studied for years; however, several questions about the detailed mechanism of radiation damage formation remain largely unanswered. Among all biomolecules exposed to radiation, DNA plays an important role because any damage to its molecular structure can affect the whole cell and may lead to chromosomal rearrangements resulting in genomic instability or cell death. To identify and characterize damage induced in the DNA sugar-phosphate backbone, in this work we performed x-ray absorption spectroscopy at the P K-edge on DNA irradiated with either UVA light or protons. By combining the experimental results with theoretical calculations, we were able to establish the types and relative ratio of lesions produced by both UVA and protons around the phosphorus atoms in DNA.

Spore-forming bacteria that cause diseases pose a danger in our society. When in spore form, bacteria can survive high temperatures and resist a plethora of disinfection chemicals. Effective disinfection approaches are thus critical. Since a population of bacterial spores is heterogeneous in many aspects, single spore analyzing methods are suitable when heterogeneous information cannot be neglected. We present in this work a high-resolution Laser Raman optical tweezers that can trap single spores and characterize their Raman spectra. We first evaluate our system by measuring Raman spectra of spores, and purified DNA and DPA. Thereafter, we expose Bacillus thuringiensis spores to peracetic acid, chlorine dioxide, and sodium hypochlorite, which are common disinfection chemicals. The data reveals how these agents change the constitutes of a spore over time, thus improving on the mode of action of these disinfection chemicals.

I det föreliggande arbetet undersöks fragmenteringen av en joniserad molekyl Cystin, som simulerats medelst molekyldynamik och kvantmekanik. Cystin betraktas som ett modellsystem för större peptidstrukturer -- något som i längden kan bidra till större förståelse för fotofragmentering av proteiner, vilket i sin tur är avgörande inom nya metoder för strukturbestämning. Analysprogrammet skrevs i Python och delvis i samarbete med en annan student. Molekylens fotofragmentering analyseras med avseende på bindningsintegritet över tid, samt mass-laddningskvot hos de resulterande fragmenten. I allmänhet sönderfaller molekylen till fler och mindre fragment ju högre joniseringsnivån är.

Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality. Seeding is one such optimization method. In classical seeding experiments, the seed crystals are put into new, albeit similar, conditions. The past decade has seen the emergence of an alternative seeding strategy: microseed matrix screening (MMS). In this strategy, the seed crystals are transferred into conditions unrelated to the seed source. Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups, better diffracting crystals and crystallization of previously uncrystallizable targets. MMS can be implemented robotically, making it a viable option for drug-discovery programs. In conclusion, MMS is a simple, time-and cost-efficient optimization method that is applicable to many recalcitrant crystallization problems.