The design and fabrication of solar cells have recently witnessed the exploration of non-fullerene-based acceptor molecules for higher efficiency. In this study, the optical and electronic properties of four new three-dimensional (3D) star-shaped acceptor molecules (M1, M2, M3, and M4) are evaluated for use as acceptor molecules in organic solar cells. These molecules contain a triphenylamine donor core with diketopyrrolopyrrole acceptor arms linked via a thiophene bridge unit. Molecules M1–M4 are characterized by different end-capped acceptor moieties, including 2-(5-methylene-6-oxo-5,6-dihydrocyclopenta-b-thiophen-4-ylidene)malononitrile (M1), 2-(2-methylene-3-oxo-2,3-dihydroinden-1-ylidene)malononitrile (M2), 2-(5-methyl-2-methylene-3-oxo-2,3-dihydroinden-1-ylidene)malononitrile (M3), and 3-methyl-5-methylnene-thioxothiazolidin-4-one (M4). The properties of the newly designed molecules were compared with a well-known reference compound R, which was recently reported as an excellent acceptor molecule for organic solar cells. Molecules M1–M4 exhibit suitable frontier molecular orbital patterns for charge mobility. M2 shows maximum absorption (λ_max) at 846.8 nm in dichloromethane solvent, which is ideal for the design of transparent solar cells. A strong electron withdrawing end-capped acceptor causes a red shift in absorption spectra. All molecules are excellent for hole mobility due to a lower value of λ_h compared to the reference R.

Idag räcker det med DNA från enstaka celler för att kunna få fram en DNA-profil som kan jämföras med per-soner eller andra DNA-spår. En DNA-träff mot ett biologiskt spår kan utgöra en mycket stark bevisning och vara avgörande för en fällande dom. DNA-teknik gör det möjligt att analysera och ta fram en DNA-profil för de allra flesta typer av humana biologiska spår som avsatts i samband med brott, såsom blod, sperma, vaginalsekret, saliv, hår och ”kontaktspår”. Teknikerna har med åren utvecklats och förfinats. På senare år har också det internationella utbytet av DNA-profiler ökat samtidigt som fortsatt teknik- och metodutveckling banar väg för fördjupade analy-ser som kan bidra till att klara upp brott. Det kan handla om att utifrån DNA-spår ringa in ungefärlig ålder, ursprung, hårfärg, ögonfärg och kroppsstorlek på en misstänkt gärningsman

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.

Most of the ongoing projects aimed at the development of specific therapies and vaccines against COVID-19 use the SARS-CoV-2 spike (S) protein as the main target. The binding of the spike protein with the ACE2 receptor (ACE2) of the host cell constitutes the first and key step for virus entry. During this process, the receptor binding domain (RBD) of the S protein plays an essential role, since it contains the receptor binding motif (RBM), responsible for the docking to the receptor. So far, mostly biochemical methods are being tested in order to prevent binding of the virus to ACE2. Here we show, with the help of atomistic simulations, that external electric fields of easily achievable and moderate strengths can dramatically destabilise the S protein, inducing long-lasting structural damage. One striking field-induced conformational change occurs at the level of the recognition loop L3 of the RBD where two parallel beta sheets, believed to be responsible for a high affinity to ACE2, undergo a change into an unstructured coil, which exhibits almost no binding possibilities to the ACE2 receptor. We also show that these severe structural changes upon electric-field application also occur in the mutant RBDs corresponding to the variants of concern (VOC) B.1.1.7 (UK), B.1.351 (South Africa) and P.1 (Brazil). Remarkably, while the structural flexibility of S allows the virus to improve its probability of entering the cell, it is also the origin of the surprising vulnerability of S upon application of electric fields of strengths at least two orders of magnitude smaller than those required for damaging most proteins. Our findings suggest the existence of a clean physical method to weaken the SARS-CoV-2 virus without further biochemical processing. Moreover, the effect could be used for infection prevention purposes and also to develop technologies for in-vitro structural manipulation of S. Since the method is largely unspecific, it can be suitable for application to other mutations in S, to other proteins of SARS-CoV-2 and in general to membrane proteins of other virus types.

Molecular tools for fluorescent imaging of protein aggregates are essential for understanding the significance of these pathological hallmarks in proteopathic neurodegenerative diseases, such as Alzheimers disease. Here, we report the synthesis of a series of oligothiophene porphyrin hybrids, OTPHs, and the evaluation of these dyes for fluorescent imaging of beta-amyloid aggregates in tissue sections from a transgenic mouse model with Alzheimers disease pathology. The OTPHs proved to be successful for spectral and lifetime imaging assessment of protein deposits and our findings confirm that the enhanced spectral range and distinct lifetime diversity of these novel tools allow a more precise assessment of heterogeneous amyloid morphology compared with the corresponding oligothiophene dye. In addition, the chemical identity of the porphyrin moiety, as well as the spacing between the two optical active moieties, influenced the OTPHs performance for fluorescent assignment of the protein deposits. We foresee that our findings will aid in the chemical design of dyes that can be utilized as optical tools for studying the polymorphic nature of protein aggregates associated with proteopathic neurodegenerative diseases.

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.

Biomaterials are materials that are specifically designed to be in contact with biological systems and have for a long time been used in medicine. Examples of biomaterials range from sophisticated prostheses used for replacing outworn body parts to ordinary contact lenses. Currently it is possible to create biomaterials that can e.g. specifically interact with cells or respond to certain stimuli. Peptides, the shorter version of proteins, are excellent molecules for fabrication of such biomaterials. By following and developing design rules it is possible to obtain peptides that can self-assemble into well-defined nanostructures and biomaterials.

The aim of this thesis is to create ”smart” and tunable biomaterials by molecular self-assembly using dimerizing –helical polypeptides. Two different, but structurally related, polypeptide-systems have been used in this thesis. The EKIV-polypeptide system was developed in this thesis and consists of four 28-residue polypeptides that can be mixed-and-matched to self-assemble into four different coiled coil heterodimers. The dissociation constant of the different heterodimers range from μM to < nM. Due to the large difference in affinities, the polypeptides are prone to thermodynamic social self-sorting. The JR-polypeptide system, on the other hand, consists of several 42-residue de novo designed helix-loop-helix polypeptides that can dimerize into four-helix bundles. In this work, primarily the glutamic acid-rich polypeptide JR2E has been explored as a component in supramolecular materials. Dimerization was induced by exposing the polypeptide to either Zn²⁺, acidic conditions or the complementary polypeptide JR2K.

By conjugating JR2E to hyaluronic acid and the EKIV-polypeptides to star-shaped poly(ethylene glycol), respectively, highly tunable hydrogels that can be self-assembled in a modular fashion have been created. In addition, self-assembly of spherical superstructures has been investigated and were obtained by linking two thiol-modified JR2E polypeptides via a disulfide bridge in the loop region. ŒThe thesis also demonstrates that the polypeptides and the polypeptide-hybrids can be used for encapsulation and release of molecules and nanoparticles. In addition, some of the hydrogels have been explored for 3D cell culture. By using supramolecular interactions combined with bio-orthogonal covalent crosslinking reactions, hydrogels were obtained that enabled facile encapsulation of cells that retained high viability.

The results of the work presented in this thesis show that dimerizing α–helical polypeptides can be used to create modular biomaterials with properties that can be tuned by specific molecular interactions. The modularity and the tunable properties of these smart biomaterials are conceptually very interesting andmake them useful in many emerging biomedical applications, such as 3D cell culture, cell therapy, and drug delivery

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Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62-160 nm. Such nanoparticles have a negative surface charge (-11 to -18 mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine (TM) 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.

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.

Minimal absent words (MAW) of a genomic sequence are subsequences that are absent themselves but the subwords of which are all present in the sequence. The characteristic distribution of genomic MAWs as a function of their length has been observed to be qualitatively similar for all living organisms, the bulk being rather short, and only relatively few being long. It has been an open issue whether the reason behind this phenomenon is statistical or reflects a biological mechanism, and what biological information is contained in absent words. % In this work we demonstrate that the bulk can be described by a probabilistic model of sampling words from random sequences, while the tail of long MAWs is of biological origin. We introduce the novel concept of a core of a minimal absent word, which are sequences present in the genome and closest to a given MAW. We show that in bacteria and yeast the cores of the longest MAWs, which exist in two or more copies, are located in highly conserved regions the most prominent example being ribosomal RNAs (rRNAs). We also show that while the distribution of the cores of long MAWs is roughly uniform over these genomes on a coarse-grained level, on a more detailed level it is strongly enhanced in 3' untranslated regions (UTRs) and, to a lesser extent, also in 5' UTRs. This indicates that MAWs and associated MAW cores correspond to fine-tuned evolutionary relationships, and suggest that they can be more widely used as markers for genomic complexity.

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.

Aim of present study was to synthesize a novel chitosan-quercetin (CTS-QT) complex by making a carbodiimide linkage using maleic anhydride as cross-linker and to investigate its enhanced antibacterial and antioxidant activities as compare to pure CTS and QT. Equimolar concentration of QT and maleic anhydride were used to react with 100 mg CTS to form CTS-QT complex. For this purpose, three bacterial strains namely E. Coli, S. Aureus and P. Aeruginosa were used for in-vitro antibacterial analysis (ZOI, MIC, MBC, checker board and time kill assay). Later molecular docking studies were performed on protein structure of E. Coli to assess binding affinity of pure QT and CTS-QT complex. MD simulations with accelerated settings were used to explore the protein-ligand complexs binding interactions and stability. Antioxidant profile was determined by performing DPPH center dot radical scavenging assay, total antioxidant capacity (TAC) and total reducing power (TRP) assays. Delivery mechanism to CTS-QT complex was improved by synthesizing polycaprolactone containing microspheres (CTS-QT-PCL-Levo-Ms) using Levofloxacin as model drug to enhance their antibacterial profile. Resulted microspheres were evaluated by particle size, charge, surface morphology, in-vitro drug release and hemolytic profile and are all were found within limits. Antibacterial assay revealed that CTS-QT-PCL-Levo-Ms showed more than two folds increased bactericidal activity against E. Coli and P. Aeruginosa, while 1.5 folds against S. Aureus. Green colored formation of phosphate molybdate complexes with highest 85 +/- 1.32% TAC confirmed its antioxidant properties. Furthermore, molecular docking and dynamics studies revealed that CTS-QT was embedded nicely within the active pocket of UPPS with binding energy greater than QT with RSMD value of below 1.5. Conclusively, use of maleic acid, in-vitro and in-silico antimicrobial studies confirm the emergence of CTS-QT complex containing microspheres as novel treatment strategy for all types of bacterial infections. Communicated by Ramaswamy H. Sarma

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.

NEK7 is a NIMA related-protein kinase that plays a crucial role in spindle assembly and cell division. Dysregulation of NEK7 protein leads to development and progression of different types of malignancies including colon and breast cancers. Therefore, NEK7 could be considered as an attractive target for anti-cancer drug discovery. However, few efforts have been made for the development of selective inhibitors of NIMA-related kinase but still no FDA approved drug is known to selectively inhibit the NEK7 protein. Dacomitinib and Neratinib are two Enamide derivatives that were approved for treatment against non-small cell lung cancer and breast cancer respectively. Drug repurposing is a time and cost-efficient method for re-evaluating the activities of previously authorized medications. Thus, the present research involves the repurposing of two FDA-approved medications via comprehensive in silico approach including Density functional theory (DFTs) studies which were conducted to determine the electronic properties of the Dacomitinib and Neratinib. Afterward, binding orientation of selected drugs inside NEK7 activation loop was evaluated through molecular docking approach. Selected drugs exhibited potential molecular interactions engaging important amino acid residues of active site. The docking score of Dacomitinib and Neratinib was -30.77 and -26.78 kJ/mol, respectively. The top ranked pose obtained from molecular docking was subjected to Molecular Dynamics (MD) Simulations for investigating the stability of protein-ligand complex. The RMSD pattern revealed the stability of protein-ligand complex throughout simulated trajectory. In conclusion, both drugs displayed inhibitory efficacy against NEK7 protein and provide a prospective therapy option for malignant malignancies linked with NEK7. Communicated by Ramaswamy H. Sarma

Significant metabolic pathways have been linked to AKR1B1 and AKR1B10. These enzymes are crucial biological targets in the therapy of colon cancer. In the past several decades, drug repurposing has gained appeal as a time and cost-efficient strategy for providing new indications for existing drugs. The structural properties of the plant-based alkaloidal drugs theobromine and theophylline were examined using density functional theory (DFT) computations, where the B3LYP/SVP method was used to quantify the dipole moment, polarizability, and optimization energy. Optimized structures obtained through DFT studies were docked inside the active pocket of target proteins to evaluate their inhibitory potential. Moreover, molecular dynamic simulation provides significant insight into a dynamic view of molecular interactions. The findings of current revealed theobromine and theophylline as strong AKR1B1 and AKR1B10 inhibitors, respectively. In addition, the anti-cancer potential of theophylline and theobromine was validated by targeting various tumor proteins, i.e. NF-kappa B, cellular tumor antigen P53 and caspase-3 using a molecular docking approach. Theobromine was found to be strongly interacted with NF-kappa B and caspase-3, whereas theophylline potentially inhibited cellular tumor antigen P53. In addition, the ADMET characteristics of theobromine and theophylline were identified, confirming their drug-like capabilities. These results should open the way for further experimental validation and structure-based drug design/repurposing of AKR1B1/AKR1B10 inhibitors for the treatment of colon cancer and associated malignancies. Communicated by Ramaswamy H. Sarma

We describe a new way to calculate the electrostatic properties of macromolecules which eliminates the assumption of a constant dielectric value in the solvent region, resulting in a Generalized Poisson-Boltzmann-Langevin equation (GPBLE). We have implemented a web server (http://lorentz.immstr.pasteur.fr/pdb_hydro.php) that both numerically solves this equation and uses the resulting water density profiles to place water molecules at preferred sites of hydration. Surface atoms with high or low hydration preference can be easily displayed using a simple PyMol script, allowing for the tentative prediction of the dimerization interface in homodimeric proteins, or lipid binding regions in membrane proteins. The web site includes options that permit mutations in the sequence as well as reconstruction of missing side chain and/or main chain atoms. These tools are accessible independently from the electrostatics calculation, and can be used for other modeling purposes. We expect this web server to be useful to structural biologists, as the knowledge of solvent density should prove useful to get better fits at low resolution for X-ray diffraction data and to computational biologists, for whom these profiles could improve the calculation of interaction energies in water between ligands and receptors in docking simulations.

Here, we study and compare the mechanisms of action of the GroEL/GroES and the TRiC chaperonin systems on MreB client protein variants extracted from E. coli. MreB is a homologue to actin in prokaryotes. Single-molecule fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence polarization anisotropy report the binding interaction of folding MreB with GroEL, GroES and TRiC. Fluorescence resonance energy transfer (FRET) measurements on MreB variants quantified molecular distance changes occurring during conformational rearrangements within folding MreB bound to chaperonins. We observed that the MreB structure is rearranged by a binding-induced expansion mechanism in TRiC, GroEL and GroES. These results are quantitatively comparable to the structural rearrangements found during the interaction of beta-actin with GroEL and TRiC, indicating that the mechanism of chaperonins is conserved during evolution. The chaperonin-bound MreB is also significantly compacted after addition of AMP-PNP for both the GroEL/ES and TRiC systems. Most importantly, our results showed that GroES may act as an unfoldase by inducing a dramatic initial expansion of MreB (even more than for GroEL) implicating a role for MreB folding, allowing us to suggest a delivery mechanism for GroES to GroEL in prokaryotes.

Plants and other green organisms harvest sunlight by green chlorophyll pigments and covertit to chemical energy (sugars) and oxygen in a process called photosynthesis providing the foundation for life on Earth. Although it is unanimously believed that oceanic phytoplanktons are the main contributors to the global photosynthesis, the contribution of coniferous boreal forests distributed across vast regions of the northern hemisphere cannot be undermined. Hence boreal forests account signifificantly for social, economical and environmental sustainability. Not only do conifers thrive in the tundra regions with extreme climate, but they also maintain their needles green over the boreal winter. A question remains; what makes them so resilient? In this respect, we aimed to understand the remarkable winter adaptation strategies in two dominant boreal coniferous species,i.e., Pinus sylvestris and Picea abies. First, we mapped the transcriptional landscape in Norway spruce (Picea abies) needles over the annual cycle. Transcriptional changes in the nascent needles reflflected a sequence of developmental processes and active vegetative growth during early summer and summer. Later after maturation, transcriptome reflflected activated defense against biotic factors and acclimationin response to abiotic environmental cues such as freezing temperatures during winter. Secondly, by monitoring the photosynthetic performance of Scot pine needles, we found that the trees face extreme stress during the early spring (Feb-Mar) when sub-zero temperatures are accompanied by high solar radiation. At this time, drastic changes occur in the thylakoid membranes of the chloroplast that allows the mixing of photosystem I and photosystem II that typically remain laterally segregated. This triggers direct energy transfer from PSII to PSI and thus protects PSII from damage. Furthermore, we found that this loss of lateral segregation may be a consequence of triple phosphorylationof Lhcb1 (Light harvesting complex1 of photosystem II). The structural changes in thylakoid membranes also lead to changes inthe thylakoid macro domain organisationand pigment protein composition. Furthermore, we discovered that while PSII is protected by direct energy transfer, the protection of PSI is provided through photoreduction of oxygen by flavodiiron proteins, which in turn allows P700 to stay in an oxidised state necessary for direct energy transfer. These coordinated cascades of changes concomitantly protect both PSI and PSII to maintain the needles green over the winter.

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.

The effects of low doses of ionizing radiation on cellular development in the nervous system are presently unclear. The focus of the present study was to examine low-dose gamma-radiation-induced effects on the differentiation of neuronal cells and on the development of neural stem cells to glial cells. Human neuroblastoma SH-SY5Y cells were exposed to (137)Cs gamma rays at different stages of retinoic acid-induced neuronal differentiation, and neurite formation was determined 6 days after exposure. When SH-SY5Y cells were exposed to low-dose-rate gamma rays at the onset of differentiation, the number of neurites formed per cell was significantly less after exposure to either 10, 30 or 100 mGy compared to control cells. Exposure to 10 and 30 mGy attenuated differentiation of immature C17.2 mouse-derived neural stem cells to glial cells, as verified by the diminished expression of glial fibrillary acidic protein. Proteomic analysis of the neuroblastoma cells by 2D-PAGE after 30 mGy irradiation showed that proteins involved in neuronal development were downregulated. Proteins involved in cell cycle and proliferation were altered in both cell lines after exposure to 30 mGy; however, the rate of cell proliferation was not affected in the low-dose range. The radiation-induced attenuation of differentiation and the persistent changes in protein expression is indicative of an epigenetic rather than a cytotoxic mechanism. (C) 2011 by Radiation Research Society

Escherichia coli express adhesion pili that mediate attachment to host cell surfaces and are exposed to body fluids in the urinary and gastrointestinal tracts. Pilin subunits are organized into helical polymers, with a tip adhesin for specific host binding. Pili can elastically unwind when exposed to fluid flow forces, reducing the adhesin load, thereby facilitating sustained attachment. Here we investigate biophysical and structural differences of pili commonly expressed on bacteria that inhabit the urinary and intestinal tracts. Optical tweezers measurements reveal that Class 1a pili of uropathogenic E. coli (UPEC), as well as Class 1b of enterotoxigenic E. coli (ETEC), undergo an additional conformational change beyond pilus unwinding, providing significantly more elasticity to their structure than ETEC Class 5 pili. Examining structural and steered molecular dynamics simulation data, we find this difference in Class 1 pili subunit behavior originates from an alpha-helical motif that can unfold when exposed to force. A disulfide bond cross-linking beta-strands in Class 1 pili stabilizes subunits, allowing them to tolerate higher forces than Class 5 pili that lack this covalent bond. We suggest that these extra contributions to pilus resiliency are relevant for the UPEC niche since resident bacteria are exposed to stronger, more transient drag forces compared to those experienced by ETEC bacteria in the mucosa of the intestinal tract. Interestingly, Class 1b ETEC pili include the same structural features seen in UPEC pili, while requiring lower unwinding forces that are more similar to those of Class 5 ETEC pili.

Bacterial adhesion pili are essential virulence factors for many pathogenic Escherichia coli, including bacteria that cause urinary tract infections (UPEC) and diarrheal diseases (ETEC). To sustain adhesion under forces similar to those in the fluid environments of the urinary tract and gastrointestinal tract, these pili (also called fimbriae) can extend to over seven times their original length. Both UPEC and ETEC can uncoil their quaternary structure under pulling force and re-coil to their helical form when the force is reduced, as observed using optical tweezers. However, after extension to a linear polymer UPEC undergo an additional reversible conformational change, that is not seen in ETEC. The mechanism for this conformational change in UPEC is not known. Therefore, to obtain a comprehensive picture of pilus extension we have taken a synergistic approach that combines optical tweezer experiments, structural data from cryo-EM, and steered molecular dynamics simulations to investigate the response of pilin subunits to force.

Our multi-faceted approach provides novel molecular-scale insights into the structural changes that occur in UPEC and ETEC pili under pulling forces. We find that the conformational change observed in UPEC pili in optical tweezer experiments is correlated with the presence of an alpha helix. In addition, structural analysis and steered molecular dynamics simulations show that there is a disulfide bond that provides additional stability of UPEC pilin subunits that is not observed in ETEC pilins, which lack cysteine residues. Together, these results suggest that the mechanism of extension of bacterial adhesion pili is related to their environmental niche, and the magnitude of fluid forces in the urinary tract versus the GI tract.

An important step in the production of inflammatory mediators of the leukotriene family is the Ca2+ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca2+ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron micros-copy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane.

Biophysical studies with myosin motor fragments (heavy meromyosin; HMM and subfragment 1; S1) adsorbed to artificial surfaces, are important for elucidation of actomyosin function. In spite of the widespread use of such in vitro motility assays and single molecule studies, little is known about the adsorption geometry and effects of protein-surface interactions on the motor properties. Here, we investigate these factors with focus on HMM using quartz crystal microbalance with dissipation (QCM-D) and total internal reflection fluorescence (TIRF) spectroscopy based ATPase assays. In the latter, we monitored the turnover of Alexa-fluor647-ATP (Alexa-ATP) by surface adsorbed HMM. Studies were performed with HMM/S1 adsorbed to model hydrophilic (SiO2) or hydrophobic (trimethyl-chlorosilane [TMCS] - derivatized) surfaces. The results suggest that adsorption of HMM is weakened on SiO2 (but not on TMCS) at high (245 mM) compared to low (65 mM) ionic strengths. The changes in ionic strength were also associated with structural changes in the protein layer according to QCM-D studies. Moreover, the TIRF based ATPase assay suggested a larger fraction of HMM molecules with low catalytic activity on SiO2. These and other TIRF and QCM-D results, suggest that HMM preferentially adsorbs to negatively charged hydrophilic surfaces via the actin-binding region. In contrast, the majority of the HMM molecules seem to adsorb via their C-terminal tail on moderately hydrophobic surfaces. In the latter case the catalytic sites appear to be close to, but not immobilized on the surface. The results with HMM were compared to, and found consistent with, QCM-D and TIRF-data obtained with S1 motor fragments.

Modelling ionic channels represents a fundamental step towards developing biologically detailed neuron models. Until recently, the voltage-gated ion channels have been mainly modelled according to the formalism introduced by the seminal works of Hodgkin and Huxley (HH). However, following the continuing achievements in the biophysical and molecular comprehension of these pore-forming transmembrane proteins, the HH formalism turned out to carry limitations and inconsistencies in reproducing the ion-channels electrophysiological behaviour. At the same time, Markov-type kinetic models have been increasingly proven to successfully replicate both the electrophysiological and biophysical features of different ion channels. However, in order to model even the finest non-conducting molecular conformational change, they are often equipped with a considerable number of states and related transitions, which make them computationally heavy and less suitable for implementation in conductance-based neurons and large networks of those. In this purely modelling study we develop a Markov-type kinetic model for all human voltage-gated sodium channels (VGSCs). The model framework is detailed, unifying (i.e., it accounts for all ion-channel isoforms) and computationally efficient (i.e. with a minimal set of states and transitions). The electrophysiological data to be modelled are gathered from previously published studies on whole-cell patch-clamp experiments in mammalian cell lines heterologously expressing the human VGSC subtypes (from NaV1.1 to NaV1.9). By adopting a minimum sequence of states, and using the same state diagram for all the distinct isoforms, the model ensures the lightest computational load when used in neuron models and neural networks of increasing complexity. The transitions between the states are described by original ordinary differential equations, which represent the rate of the state transitions as a function of voltage (i.e., membrane potential). The kinetic model, developed in the NEURON simulation environment, appears to be the simplest and most parsimonious way for a detailed phenomenological description of the human VGSCs electrophysiological behaviour.

Virus entry is a complex dynamic multistep process requiring a series of fine-tuned events mediating virus diffusion through the glycocalyx, its attachment to the cell membrane and lateral diffusion to the point of entry. A number of enveloped viruses, including herpes simplex viruses (HSV) attach to susceptible host cells via interaction between their glycoproteins and cell-surface glycosaminoglycans (GAGs). In our work, we study the molecular and physical mechanisms modulating HSV binding, diffusion and release from cell-surface glycosaminoglycans. Using single virus tracking in combination with either in vitro minimal models of the cell surface or live cell microscopy, we gain insights into the modulatory function of protein glycosylation (the presence of mucin-like regions on viral glycoproteins) and interrogate the role of GAG sulfation in the process. We show that mucin-like regions found on the glycoproteins of HSV-1 and HSV-2 play an important role in modulating the interaction, an observation further supported by cell experiments. We further show that the diffusion of virions on the surface depends on the type of GAGs and their degree of sulfation. Taken together, our research contributes to a better understanding of the mechanisms underlying the interaction between a virus and the surface of its host. Such insights will without doubt facilitate the design of more efficient antiviral drugs or vaccines.

The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.

Lipid assemblies have attracted considerable interest as components in bioanalytical sensors. They provide a native-like environment for the immobilization of membrane proteins and for the study of membrane-related processes. Liposomes are also excellent bioanalytical assay components since selected functionalities can be added to the membrane while their aqueous interior can encapsulate a variety of molecules. This chapter highlights the potential of lipid assemblies in surface-based affinity sensors. It first describes how such sensors are created, providing an overview of lipid immobilization strategies together with a summary of the major transduction techniques used to probe binding at and transport through membrane interfaces. It then reviews the implementation of lipid-based sensors in the study of membrane proteins and membrane-mediated interactions, followed by a discussion of the potential of liposomes as nanoscale labels and as nanoreactors. Finally, it illustrates how external forces can be used to manipulate membrane component for biosensing applications.

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.

Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold – a substrate commonly used in nanotechnology – and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct adsorption behavior of the deoxyribonucleotides (i.e., a nitrogenous base, a deoxyribose sugar, and a phosphate group) and on the factors that govern the DNA–gold bond strength. Here, using single molecule force spectroscopy, we investigated the interaction of the four individual nucleotides, adenine, guanine, cytosine, and thymine, with gold. Experiments were performed in three salinity conditions and two surface dwell times to reveal the factors that influence nucleotide–Au bond strength. Force data show that, at physiological ionic strength, adenine–Au interactions are stronger, asymmetrical and independent of surface dwell time as compared to cytosine–Au and guanine–Au interactions. We suggest that in these conditions only adenine is able to chemisorb on gold. A decrease of the ionic strength significantly increases the bond strength for all nucleotides. We show that moderate ionic strength along with longer surface dwell period suggest weak chemisorption also for cytosine and guanine.

The extracellular polysaccharide hyaluronan (HA) is ubiquitous in all vertebrate tissues, where its various functions are encoded in the supramolecular complexes and matrices that it forms with HA-binding proteins (hyaladherins). In tissues, these supramolecular architectures are frequently subjected to mechanical stress, yet how this affects the intermolecular bonding is largely unknown. Here, we used a recently developed single-molecule force spectroscopy platform to analyze and compare the mechanical strength of bonds between HA and a panel of hyaladherins from the Link module superfamily, namely the complex of the proteoglycan aggrecan and cartilage link protein, the proteoglycan versican, the inflammation-associated protein TSG-6, the HA receptor for endocytosis (stabilin-2/HARE), and the HA receptor CD44. We find that the resistance to tensile stress for these hyaladherins correlates with the size of the HA-binding domain. The lowest mean rupture forces are observed for members of the type A subgroup (i.e., with the shortest HA-binding domains; TSG-6 and HARE). In contrast, the mechanical stability of the bond formed by aggrecan in complex with cartilage link protein (two members of the type C subgroup, i.e., with the longest HA-binding domains) and HA is equal or even superior to the high affinity streptavidin⋅biotin bond. Implications for the molecular mechanism of unbinding of HA⋅hyaladherin bonds under force are discussed, which underpin the mechanical properties of HA⋅hyaladherin complexes and HA-rich extracellular matrices.

Air-filled polyvinyl alcohol microbubbles (PVA-MBs) were recently introduced as a contrast agent for ultrasound imaging. In the present study, we explore the possibility of extending their application in multimodal imaging by labeling them with a near infrared (NIR) fluorophore, VivoTag-680. PVA-MBs were injected intravenously into FVB/N female mice and their dynamic biodistribution over 24 h was determined by 3D-fluorescence imaging co-registered with 3D-mu CT imaging, to verify the anatomic location. To further confirm the biodistribution results from in vivo imaging, organs were removed and examined histologically using bright field and fluorescence microscopy. Fluorescence imaging detected PVA-MB accumulation in the lungs within the first 30 min post-injection. Redistribution to a low extent was observed in liver and kidneys at 4 h, and to a high extent mainly in the liver and spleen at 24 h. Histology confirmed PVA-MB localization in lung capillaries and macrophages. In the liver, they were associated with Kupffer cells; in the spleen, they were located mostly within the marginal-zone. Occasional MBs were observed in the kidney glomeruli and interstitium. The potential application of PVA-MBs as a contrast agent was also studied using ultrasound (US) imaging in subcutaneous and orthotopic pancreatic cancer mouse models, to visualize blood flow within the tumor mass. In conclusion, this study showed that PVA-MBs are useful as a contrast agent for multimodal imaging. (C) 2015 Elsevier Inc. All rights reserved.

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.

Since the 1980s, cremation has become the fastest growing area of the U.S. funeral industry. At the same time, the number of litigations against funeral homes and cremation facilities has increased. Forensic anthropologists are often asked to determine whether the contents of an urn are actually cremated bone, and to address questions regarding the identity of the remains. This study uses both metric and chemical analyses for resolving a case of contested cremains. A cremains weight of 2021.8 g was predicted based on the decedent's reported stature and weight. However, the urn contents weighed 4173.5 g. The urn contents also contained material inconsistent with cremains (e.g., moist sediment, stones, ferrous metal). Analysis using XRD and SEM demonstrated that the urn contained thermally altered bone as well as inorganic material consistent with glass fiber cement. Although forensically challenging, cremains cases such as this one can be resolved using a multidisciplinary approach.

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.

Specific interactions that depend on the nature of electrolytes are observed when proteins and other molecules are studied by potentiometric, spectroscopic and theoretical methods at high salt concentrations. More recently, it became clear that such interactions may also be observed in solutions that can be described by the Debye-Hückel theory, i.e., at physiological (0.1 mol dm⁻³) and lower concentrations. We carried out molecular dynamics simulations of bovine serum albumin in physiological solutions at T=300 and 350 K. Analysis of the simulations revealed some differences between LiCl solutions and those of NaCl and KCl. The binding of Li⁺ ions to the protein was associated with a negative free energy of interaction whereas much fewer Na⁺ and K⁺ ions were associated with the protein surface. Interestingly, unlike other proteins BSA does not show a preference to Na⁺ over K⁺. Quantum chemical calculations identified a significant contribution from polarisation to the hydration of Li⁺ and (to a lesser degree) Na⁺, which may indicate that polarisable force-fields will provide more accurate results for such systems.

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 persistence length (LP) of a polymer is proportional to its flexural rigidity and quantifies the decay length of its tangent angle (for a polymer freely suspended in solution). Further, it has been suggested that the decay length for the sliding direction of heavy meromyosin (HMM) propelled actin filaments in the in vitro motility assay (IVMA) is quantitatively identical to Lp of the free leading filament end. On this assumption we measured LP under different conditions to address a hypothesis that the actin filament exists in different metastable conformations, each characterized by a different flexural rigidity. The following values for Lp (mean 5 95 % confidence limits) were obtained: 1. with phalloidin (Ph) in solution: 12.61 5 0.65 mm (N=809). 2. without Phin solution: 9.07 5 1.06 mm (N=811), 3. with Ph and HMM in solution (rigor):10.21 5 0.75 mm (N=429), 4. without Ph (from IVMA paths; 1 mM MgATP):10.0850.66 mm (N=309), 5. with Ph, IVMA (1 mM MgATP): 11.41 5 0.57 mm (N=243), 6. with Ph, IVMA, 0.05 mM MgATP: 6.30 5 0.27 mm (N=383) and 7. without Ph, IVMA, 0.02-0.05 mM MgATP: 5.33 5 0.37 mm (N=161). The re-sults are consistent with different actin filament states where one is stabilized by phalloidin and one is favored by HMM binding and the absence of Ph. Effects of HMM are consistent with a possible role of the structural state of actin filaments in effective actomyosin motility. The very low LP found for IVMA at low [MgATP] (6-7) may reflect the presence of an actin filament state populated at low average cross-bridge strains, possibly with MgADP at the active site. Alternatively, it may be due to sideways forces produced by increased number of HMM-actin interactions close to the leading filament end.