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To study the implications of highly space-demanding organic moieties on the properties of self-assembled monolayers (SAMs), triptycyl thiolates and selenolates with and without methylene spacers on Au(111) surfaces were comprehensively studied using ultra-high vacuum infrared reflection absorption spectroscopy, X-ray photoelectron spectroscopy, near-edge X-ray absorption fine structure spectroscopy and thermal desorption spectroscopy. Due to packing effects, the molecules in all monolayers are substantially tilted. In the presence of a methylene spacer the tilt is slightly less pronounced. The selenolate monolayers exhibit smaller defect densities and therefore are more densely packed than their thiolate analogues. The Se–Au binding energy in the investigated SAMs was found to be higher than the S–Au binding energy.
This thesis is concerned with quantum dynamical propagation methods suitable for high-dimensional systems, and their application to excitation energy transfer (EET), electron transfer (ET), and intra-molecular vibrational redistribution (IVR) in molecular aggregates. The theoretical description of these processes, which are often ultrafast – with time scales in the range of femtoseconds to picoseconds – is challenging, both with regard to quantum dynamical simulations and electronic structure calculations.
The present thesis comprises two parts. The first part concerns the implementation of a novel quantum dynamical method based on Gaussian Wavepackets (GWPs): the 2-Layer Gaussian-MCTDH (2L-GMCTDH) method. This method, which has recently been proposed in [S. Römer, M. Ruckenbauer, I. Burghardt, The Journal of Chemical Physics, 2013, 138, 064106] was implemented in a Fortran90 code and applied to various high-dimensional test systems. The second part of the thesis addresses the combined electronic structure and dynamical study of a novel type of donor-acceptor systems that have been investigated in a joint project with experimental collaboration partners at Strasbourg University. In both parts, numerical applications focus on high-dimensional model Hamiltonians for EET and ET processes.
Regarding the first part, the interest of using GWP-based methods is two-fold: First, GWPs represent spatially localized basis sets that are useful for on-the-fly dynamics in conjunction with electronic structure calculations. Second, they are naturally suited for the explicit representation of quantum mechanical system-bath type problems where a large number of vibrational bath modes are weakly perturbed from equilibrium. In this context, various methods exist that are based upon classically evolving GWP bases. A major improvement results from variational methods which involve optimized, non-classical GWP trajectories. In particular, the variational Gaussian-based Multi-Configuration Time-Dependent Hartree (GMCTDH) and its variational Multi-Configurational Gaussians (vMCG) variant were originally derived as semiclassical variants of the Multi-Configuration Time-Dependent Hartree (MCTDH) method. However, the G-MCTDH and vMCG methods mostly use Frozen Gaussian (FG) basis sets that are far less flexible than the single-particle (SPF) representation of standard MCTDH. As a consequence, a significantly larger number of GWPs are generally required to reach convergence. To remedy the lack of flexibility of the FG basis sets, the abovementioned two-layer (2L-G-MCTDH) approach has been introduced: Here, the first layer is composed of flexible SPFs, while the second layer is composed of low-dimensional FGs. The numerical scaling properties are significantly improved as compared with the conventional G-MCTDH and vMCG schemes. The first implementation of the method in an in-house Fortran90 code is presented, along with applications to (i) a model of site-to-site vibrational energy flow in the presence of intra-site vibrational energy redistribution (IVR) and (ii) a multidimensional donor-acceptor electron transfer system described within a linear vibronic coupling model. The second system relates to a model for ET at an oligothiophene-fullerene interface relevant to organic photovoltaics. Besides the description of the implementation, a detailed assessment of the convergence properties and comparison with multi-layer MCTDH (ML-MCTDH) benchmark calculations is presented. Finally, a perspective is given on the future combination with the existing ML-MCTDH scheme; indeed, such a combination is straightforward since the first layer of the 2L-G-MCTDH approach can be chosen to be orthogonal.
Regarding the second part of the thesis, two generations of a novel donor-acceptor (DA) system for organic photovoltaics applications, involving self-assembled block co-oligomers DA dyads and triads with perylene-diimide (PDI) accepter units, are addressed within a collaborative project with S. Haacke and S. Mery (University of Strasbourg). Based upon detailed excited-state electronic structure investigations along with quantum dynamical and kinetic studies, the relevant ET formation and recombination steps are characterized quantitatively, in view of optimizing the chemical design and reducing recombination losses.
In a first-generation variant of the abovementioned DA systems, which involves liquid-crystalline triads, we were able to show that a highly efficient inter-chain ET process prevails over intra-molecular ET, leading to fast recombination. Due to the latter, this system turns out to be inefficient for photovoltaic applications. To fully understand the elementary steps, high-dimensional quantum dynamics simulations were carried out using the ML-MCTDH method, in collaboration with Matthias Polkehn from our group. In the second-generation variant, which is in the focus of the present thesis, both the nanomorphology and the chemical design were modified. The present work, focuses upon the aspect of chemical design, by characterizing a series of modified DA’s, with donor units of varying length while the PDI accepter units remain unchanged. The intra-molecular ET is observed in these systems, but the processes are comparatively slow, of the order of tens to hundreds of picoseconds. Hence, a kinetic analysis using the Marcus-Levich-Jortner rate theory is employed. Among the main results of the study is that addition of an electron donating amine unit strongly increases the lifetime of the charge-separated state, and therefore reduced recombination losses.
Overall, the present thesis shows how a combination of high-dimensional quantum dynamics, electronic structure calculations, and vibronic coupling model Hamiltonians can be employed to obtain an accurate picture of EET, ET, and IVR in high-dimensional molecular assemblies. Furthermore, the 2L-GMCTDH method paves the way for accurate and efficient on-the-fly calculations; a suitable set-up for such calculations is currently in progress.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition of abnormal heart rhythm (arrhythmia), induced by physical activity or stress. Mutations in ryanodine receptor 2 (RyR2), a Ca2+ release channel located in the sarcoplasmic reticulum (SR), or calsequestrin 2 (CASQ2), a SR Ca2+ binding protein, are linked to CPVT. For specific drug development and to study distinct arrhythmias, simple models are required to implement and analyze such mutations. Here, we introduced CPVT inducing mutations into the pharynx of Caenorhabditis elegans, which we previously established as an optogenetically paced heart model. By electrophysiology and video-microscopy, we characterized mutations in csq-1 (CASQ2 homologue) and unc-68 (RyR2 homologue). csq-1 deletion impaired pharynx function and caused missed pumps during 3.7 Hz pacing. Deletion mutants of unc-68, and in particular the point mutant UNC-68(R4743C), analogous to the established human CPVT mutant RyR2(R4497C), were unable to follow 3.7 Hz pacing, with progressive defects during long stimulus trains. The pharynx either locked in pumping at half the pacing frequency or stopped pumping altogether, possibly due to UNC-68 leakiness and/or malfunctional SR Ca2+ homeostasis. Last, we could reverse this ‘worm arrhythmia’ by the benzothiazepine S107, establishing the nematode pharynx for studying specific CPVT mutations and for drug screening.
Bacteria are highly organized organisms which are able to adapt to and propagate under a multitude of environmental conditions. Propagation hereby requires reliable chromosome replication and segregation which has to occur cooperatively with other cellular processes such as transcription, translation or signaling. Several mechanisms were proposed for segregation of the Escherichia coli (E. coli) chromosome, for example a mitotic-like active segregation model or entropy-based passive chromosome segregation. Another segregation model suggests coupled transcription, translation and insertion of membrane proteins (termed "transertion"), which links the replicating chromosome (nucleoid) to the growing cell cylinder.
Fluorescence microscopy was widely used to provide evidence for a distinct segregation model. However, the dynamic nature of bacterial chromosomes, the small bacterial size and the optical resolution limit of ~ 200-300 nm impair unveiling the underlying mechanisms. With the emergence of super-resolution fluorescence microscopy techniques and advanced labeling methods, a new toolbox became available enabling scientists to visualize biomolecules and cellular processes in unprecedented detail. Single-molecule localization microscopy (SMLM) represents a set of super-resolution microscopy techniques which relies on the temporal separation of the fluorescence signal and detection of single fluorophores. Separation can be achieved using photoactivatable or -convertible fluorescent proteins (FPs) in photoactivated localization microscopy (PALM), photoswitchable organic dyes in direct stochastic optical reconstruction microscopy (dSTORM) or dynamically binding fluorescent probes in point accumulation for imaging in nanoscale topography (PAINT). In all these techniques, the fluorescence emission pattern of single fluorophores is spatially localized with nanometer-precision. An artificial image is finally reconstructed from the coordinates of all single fluorophores detected. This provides a spatial resolution of ~ 20 nm, which is perfectly suited to investigate cellular processes in bacteria. In this thesis, different SMLM techniques were applied to study fundamental processes in E. coli. This includes determination of protein copy numbers and distributions as well as the nanoscale organization of nucleic acids and lipids.
A novel labeling approach was applied and used for super-resolution imaging of the E. coli nucleoid. It is based on the incorporation of the modified thymidine analogue 5-ethynyl-2’- deoxyuridine (EdU) into the replicating chromosome. Azide-functionalized organic fluorophores can be covalently attached to the ethynyl group of incorporated EdU bases using a copper-catalyzed "click chemistry" reaction. Under the investigated growth condition, E. coli cells exhibited overlapping replication cycles, which is commonly referred to as multi-fork replication and enables cells to divide faster than they can replicate the entire chromosome. dSTORM imaging of such labeled nucleoids revealed chromosome features with diameters of 50 - 200 nm, representing highly condensed DNA filaments. Sorting single E. coli cells by length allowed visualizing structural changes of the nucleoid throughout the cell cycle. Replicating nucleoids segregated and expanded along the bacterial long axis, while constantly covering the entire width of the cell. Measuring cell and nucleoid length revealed a relative nucleoid expansion rate of 78 ± 6 %. At the same time, nucleoids populated 63 ± 8 % of the cell length, almost exclusively being localized to the cylindrical part of the cell. This value was hence normalized to the cylindrical fraction of the cell, yielding a value of 79 ± 10 % (nucleoid-populated fraction of the cell cylinder), which is in good agreement with the observed relative nucleoid expansion rate. These results therefore support a growth-mediated segregation model, in which the chromosome is anchored to the inner membrane and passively segregated into the prospective daughter cells upon cell growth. 3-dimensional dSTORM imaging of labeled nucleoids confirmed that compacted nucleoids helically wrap along the inner membrane. Similar results were obtained by imaging orthogonally aligned E. coli cells using a holographic optical tweezer approach.
In order to visualize particular proteins together with the nucleoid, several correlative imaging workflows were established, facilitating multi-color SMLM imaging in single E. coli cells. These workflows bypass prior limitations of SMLM, including destruction of FPs by reactive oxygen species in copper-catalyzed click reactions or incompatibility of PALM imaging with dSTORM imaging buffers. A sequential SMLM imaging routine was developed which is based on postlabeling and retrieval of previously imaged cells. Optimal imaging conditions can be maintained for each fluorophore, enabling to extract quantitative information from PALM measurements while correlating the protein distribution to the nucleoid ultrastructure within the highly resolved cell envelope. Applying this workflow to an E. coli strain carrying a chromosomal rpoC - photoactivatable mCherry (PAmCh) fusion, transcribing RNA polymerase (RNAP) was found to be localized on the surface of nucleoids, where active genes are exposed towards the cytosol. During growth in nutrient-rich medium, the majority of RNAP molecules was bound to the chromosome, thus ensuring that the RNAP pool is equally distributed to the daughter cells upon cell division. This work represented the first triple-color SMLM study performed in E. coli cells. ...
Die Modulation molekularer Systeme mit Licht ist ein in den letzten Jahren immer stärker untersuchtes Forschungsgebiet. Es existiert bereits eine große Anzahl an Publikationen, die mittels statischer Spektroskopie und anderer statischer Methoden Einblicke in die ablaufenden Prozesse gewähren konnten. Untersuchungen im Ultrakurzzeitbereich sind jedoch eher selten, liefern aber detaillierte Informationen zu den ablaufenden Prozessen. Den Wissensstand diesbezüglich zu erweitern, war Ziel dieser Dissertation.
Untersucht wurden neun photoschaltbare, molekulare Dyaden hinsichtlich ihrer Dynamik nach Photoanregung. Die Dyaden setzten sich aus einem Fluorophor (Bordipyrromethen, BODIPY), einem Photoschalter (Dithienylethen, DTE; offen oder geschlossen) und gegebenenfalls einer COOH-Ankergruppe zusammen.
Die Unterschiede in den Molekülstrukturen bestanden in der Verknüpfung der einzelnen Bauteile (kurze oder lange, beziehungsweise gerade oder gewinkelte Brücke) und der Art des Fluorophors und des Photoschalters (jeweils zwei verschiedene Strukturen).
Durch Belichtung mit UV- oder sichtbarem Licht konnten photostationäre Zustände generiert werden, die 40 – 98 % geschlossenes Isomer (je nach Molekül) beziehungsweise 100 % offenes Isomer enthielten.
Unter Verwendung von Licht verschiedener Wellenlängen konnten beide Teile der Dyade (BODIPY beziehungsweise DTE) separat angeregt und hinsichtlich der ablaufenden Photodynamik untersucht werden, wobei der Fokus der Arbeit auf transienten Absorptionsmessungen mit Anregung des BODIPY lag. Bei einem Großteil der untersuchten Moleküle kam es in diesem Fall, je nach Zustand des Photoschalters, zu einem intramolekularen Energietransfer nach der Theorie von Theodor Förster. Durch diese Energietransferprozesse kommt es zu einer drastischen Verkürzung der Lebenszeit des angeregten Zustands des BODIPY. Ausgehend von Lebenszeiten im Bereich von Nanosekunden im Falle der offenen Dyaden (entspricht der Fluoreszenzlebensdauer) reduziert sich die Lebenszeit auf wenige Pikosekunden, beziehungsweise je nach Aufbau des Moleküls sogar noch weiter. Die unterschiedlich schnellen Transferprozesse sind im Sinne der Förster-Theorie durch die unterschiedlichen Entfernungen und relativen Orientierungen der beiden beteiligten Übergangsdipolmomente (von DTE und BODIPY) erklärbar.
Neben Experimenten mit Anregung des BODIPY-Teils der Dyaden wurden weitere Experimente durchgeführt, in denen der geschlossene Photoschalter direkt angeregt wurde. Aus diesen Messungen konnten Erkenntnisse über die Relaxation des DTE erlangt werden. Auf diese Weise war es möglich, bei einigen der Moleküle die Ringöffnungsreaktion zu beobachten und zu charakterisieren. Im Fall von Dyade 4 konnten zusätzlich kohärente Schwingungen des Moleküls nach Photoanregung detektiert werden, die sich anhand einer Frequenzmodulation der Absorptionsbande des BODIPY-Teils über einen Zeitbereich von 2 ps beobachten ließen.
Biophysical studies of the translation-regulating add adenine riboswitch from Vibrio vulnificus
(2017)
Bacterial gene expression can be regulated at mRNA level by cis-acting mRNA elements termed riboswitches. Riboswitches operate by conformational switching between a ligand-free and a ligand-bound state with different structures that either activate or inhibit gene expression. This PhD thesis contributes to the molecular level understanding of full-length purine riboswitches. It presents biophysical investigations on the ligand-dependent folding of the full-length translation-regulating add adenine riboswitch from the gram-negative human pathogenic marine bacterium Vibrio vulnificus (Asw). Asw has the typical bipartite riboswitch architecture with a 5’ ligand-sensing aptamer domain and a 3’ regulatory domain termed expression platform. According to the working hypothesis, Asw employs a unique thermodynamically-controlled 3-state conformational switching mechanism between an apoB, an apoA and a holo conformation to regulate translation initiation in a temperature-compensated manner. The two apo conformations are the putative translation-OFF states and the holo conformation is the putative translation-ON state of Asw. In the main project of this PhD thesis, an integrated nuclear magnetic resonance (NMR) and smFRET spectroscopic study of the full-length 112-nucleotide Asw (112Asw) was performed. The adenine-dependent folding of 112Asw was monitored at the level of base pairing interactions by NMR of the RNA imino protons, and at the level of three long-range intramolecular distances by smFRET of immobilized molecules. The integrated NMR and smFRET spectroscopic study of 112Asw yielded two major findings. First, NMR and smFRET both revealed that adenine binding to 112Asw impedes apoB formation by stabilizing the apoA secondary structure in the holo conformation without modulating tertiary structural interactions between the two riboswitch domains. This highlights the central role of competitive P1 and P4 helix formation at the interface of the aptamer and the expression platform for switching the accessibility of the ribosome binding site of 112Asw. Moreover, it strongly corroborates the hypothesis that purine riboswitches in general operate according to the key principle of a spatially decoupled secondary structural allosteric switch that proceeds without ligand-induced tertiary structural interactions between the aptamer domain and the expression platform. Second, it was uncovered by smFRET that the apoA and the holo conformation of 112Asw do not adopt a single folding state at near-physiological Mg2+ concentration. Instead, apoA and holo exhibit a persistent dynamic equilibrium between substates with an undocked (U), a short-lived docked (D1; ~s) and a Mg2+-bound long-lived docked (D2; ~10 s) aptamer kissing loop motif. In the holo conformation, the fractional population of the long-lived docked substate is ~2-fold increased compared to the apoA conformation, but undocked and docked substates are still comparably stable. The here described multiple folding states of the apoA and the holo conformation might have regulatory properties that are in between the apoB translation-OFF state and the holo-D2 translation-ON state. Additonally, an integrated NMR and smFRET analysis of 127-nucleotide Asw (127Asw) is presented. Compared to 112Asw, 127Asw is 3’-elongated by 15 nucleotides of the adenosine deaminase encoding sequence of the add gene from Vibrio vulnificus. 127Asw was chosen as mRNA template for future investigations of the interaction between Asw and the 30S ribosomal subunit. The NMR spectra of 127Asw demonstrated that 127Asw has the same overall secondary structure as 112Asw. Like for 112Asw, the combined NMR and smFRET analysis of 127Asw showed that adenine binding impedes apoB formation and stabilizes a long-lived docked aptamer kissing loop fold. However, compared to 112Asw, 127Asw has a destabilized aptamer kissing loop motif and a stabilized P4 helix in the expression platform. Finally, ligand-observed studies of the transient encounter complex between Asw and the near-cognate ligand hypoxanthine are described. By competition binding WaterLOGSY NMR experiments with hypoxanthine and the adenine analogue 2,6-diaminopurine, it could be shown that hypoxanthine binds to the same binding site of 112Asw as the cognate ligand adenine. The hypoxanthine binding constant measured with the WaterLOGSY method is in the low mM range (1.8 mM) and substantially exceeds the physiological hypoxanthine concentration in E. coli (~0.3 mM), thus ruling out that hypoxanthine binding can significantly impact the translational regulation of Asw in vivo. Also, preliminary FTIR difference spectra of 13C,15N-labelled and unlabelled hypoxanthine in complex with the pbuE adenine riboswitch aptamer and the xpt guanine riboswitch aptamer are discussed. These spectra showed a pattern of multiple IR bands that appeared to be characteristic for the respective complex.
Mistakes in translation of messenger RNA into protein are clearly a detriment to the recombinant production of pure proteins for biophysical study or the biopharmaceutical market. However, they may also provide insight into mechanistic details of the translation process. Mistakes often involve the substitution of an amino acid having an abundant codon for one having a rare codon, differing by substitution of a G base by an A base, as in the case of substitution of a lysine (AAA) for arginine (AGA). In these cases one expects the substitution frequency to depend on the relative abundances of the respective tRNAs, and thus, one might expect frequencies to be similar for all sites having the same rare codon. Here we demonstrate that, for the ADP-ribosylation factor from yeast expressed in E. coli, lysine for arginine substitutions frequencies are not the same at the 9 sites containing a rare arginine codon; mis-incorporation frequencies instead vary from less than 1 to 16%. We suggest that the context in which the codons occur (clustering of rare sites) may be responsible for the variation. The method employed to determine the frequency of mis-incorporation involves a novel mass spectrometric analysis of the products from the parallel expression of wild type and codon-optimized genes in 15N and 14N enriched media, respectively. The high sensitivity and low material requirements of the method make this a promising technology for the collection of data relevant to other mis-incorporations. The additional data could be of value in refining models for the ribosomal translation elongation process.
The p53 family of transcription factors (p53, p63 and p73) covers a wide range of functions critical for development, homeostasis and health of mammals across their lifespan. Beside the well-established tumor suppressor role, recent evidence has highlighted novel non-oncogenic functions exerted by p73. In particular, p73 is required for multiciliated cell (MCC) differentiation; MCCs have critical roles in brain and airways to move fluids across epithelial surfaces and to transport germ cells in the reproductive tract. This novel function of p73 provides a unifying cellular mechanism for the disparate inflammatory and immunological phenotypes of p73-deficient mice. Indeed, mice with Trp73 deficiency suffer from hydrocephalus, sterility and chronic respiratory tract infections due to profound defects in ciliogenesis and complete loss of mucociliary clearance since MCCs are essential for cleaning airways from inhaled pollutants, pathogens and allergens. Cross-species genomic analyses and functional rescue experiments identify TAp73 as the master transcriptional integrator of ciliogenesis, upstream of previously known central nodes. In addition, TAp73 shows a significant ability to regulate cellular metabolism and energy production through direct transcriptional regulation of several metabolic enzymes, such as glutaminase-2 and glucose-6 phosphate dehydrogenase. This recently uncovered role of TAp73 in the regulation of cellular metabolism strongly affects oxidative balance, thus potentially influencing all the biological aspects associated with p73 function, including development, homeostasis and cancer. Although through different mechanisms, p63 isoforms also contribute to regulation of cellular metabolism, thus indicating a common route used by all family members to control cell fate. At the structural level, the complexity of p73's function is further enhanced by its ability to form heterotetramers with some p63 isoforms, thus indicating the existence of an intrafamily crosstalk that determines the global outcome of p53 family function. In this review, we have tried to summarize all the recent evidence that have emerged on the novel non-oncogenic roles of p73, in an attempt to provide a unified view of the complex function of this gene within its family.
The field of dynamic nuclear polarization has undergone tremendous developments and diversification since its inception more than 6 decades ago. In this review we provide an in-depth overview of the relevant topics involved in DNP-enhanced MAS NMR spectroscopy. This includes the theoretical description of DNP mechanisms as well as of the polarization transfer pathways that can lead to a uniform or selective spreading of polarization between nuclear spins. Furthermore, we cover historical and state-of-the art aspects of dedicated instrumentation, polarizing agents, and optimization techniques for efficient MAS DNP. Finally, we present an extensive overview on applications in the fields of structural biology and materials science, which underlines that MAS DNP has moved far beyond the proof-of-concept stage and has become an important tool for research in these fields.
The transporter associated with antigen processing (TAP) selectively translocates antigenic peptides into the endoplasmic reticulum. Loading onto major histocompatibility complex class I molecules and proofreading of these bound epitopes are orchestrated within the macromolecular peptide-loading complex, which assembles on TAP. This heterodimeric ABC-binding cassette (ABC) transport complex is therefore a major component in the adaptive immune response against virally or malignantly transformed cells. Its pivotal role predestines TAP as a target for infectious diseases and malignant disorders. The development of therapies or drugs therefore requires a detailed comprehension of structure and function of this ABC transporter, but our knowledge about various aspects is still insufficient. This review highlights recent achievements on the structure and dynamics of antigenic peptides in complex with TAP. Understanding the binding mode of antigenic peptides in the TAP complex will crucially impact rational design of inhibitors, drug development, or vaccination strategies.