Integrated multiple sequence alignment

Sammeth M. Integrated multiple sequence alignment. Bielefeld (Germany): Bielefeld University; 2005.The thesis presents enhancements for automated and manual multiple sequence alignment: existing alignment algorithms are made more easily accessible and new algorithms are designed for difficult cases. Firstly, we introduce the QAlign framework, a graphical user interface for multiple sequence alignment. It comprises several state-of-the-art algorithms and supports their parameters by convenient dialogs. An alignment viewer with guided editing functionality can also highlight or print regions of the alignment. Also phylogenetic features are provided, e.g., distance-based tree reconstruction methods, corrections for multiple substitutions and a tree viewer. The modular concept and the platform-independent implementation guarantee an easy extensibility. Further, we develop a constrained version of the divide-and-conquer alignment such that it can be restricted by anchors found earlier with local alignments. It can be shown that this method shares attributes of both, local and global aligners, in the quality of results as well as in the computation time. We further modify the local alignment step to work on bipartite (or even multipartite) sets for sequences where repeats overshadow valuable sequence information. In the end a technique is established that can accurately align sequences containing eventually repeated motifs. Finally, another algorithm is presented that allows to compare tandem repeat sequences by aligning them with respect to their possible repeat histories. We describe an evolutionary model including tandem duplications and excisions, and give an exact algorithm to compare two sequences under this model

61st Annual Rocky Mountain Conference on Magnetic Resonance

Final program, abstracts, and information about the 61st annual meeting of the Rocky Mountain Conference on Magnetic Resonance, co-endorsed by the Colorado Section of the American Chemical Society and the Society for Applied Spectroscopy. Held in Copper Mountain, Colorado, July 25-29, 2022

Characterizing and Accelerating Bioinformatics Workloads on Modern Microarchitectures

Bioinformatics, the use of computer techniques to analyze biological data, has been a particularly active research field in the last two decades. Advances in this field have contributed to the collection of enormous amounts of data, and the sheer amount of available data has started to overtake the processing capability possible with current computer systems. Clearly, computer architects need to have a better understanding of how bioinformatics applications work and what kind of architectural techniques could be used to accelerate these important scientific workloads on future processors. In this dissertation, we develop a bioinformatic benchmark suite and provide a detailed characterization of these applications in common use today from a computer architect's point of view. We analyze a wide range of detailed execution characteristics including instruction mix, IPC measurements, L1 and L2 cache misses on a real architecture; and proceed to analyze the workloads' memory access characteristics. We then concentrate on accelerating a particularly computationally intensive bioinformatics workload on the novel Cell Broadband Engine multiprocessor architecture. The HMMER workload is used for protein profile searching using hidden Markov models, and most of its execution time is spent running the Viterbi algorithm. We parallelize and partition the HMMER application to implement it on the Cell Broadband Engine. In order to run the Viterbi algorithm on the 256KB local stores of the Cell BE synergistic processing units (SPEs), we present a method to develop a fast SIMD implementation of the Viterbi algorithm that reduces the storage requirements significantly. Our HMMER implementation for the Cell BE architecture, Cell-HMMER, exploits the multiple levels of parallelism inherent in this application, and can run protein profile searches up to 27.98 times faster than a modern dual-core x86 microprocessor

Purification of A-Raf and structural studies of mannitol dehydrogenase

The work herein describes the research of two separate projects: the purification of A-Raf and the crystallization and X-ray diffraction of Thermotoga maritima mannitol dehydrogenase (TmMtDH). A-Raf is a one of three Raf isoforms of serine/threonine kinases involved in the mitogen-activated protein kinase (MAPK) pathway, a cell proliferation pathway that has been associated with many cancers. In addition, only the A-Raf isoform can uniquely bind to the regulatory subunit of phosphatidylinositol-3-kinase (PI3K), which is part of the Akt/PI3K pathway and is another important signaling molecule deregulated in human cancers. Therefore, the main focus of this study was to purify and crystallize this protein in order to characterize what makes A-Raf structurally unique from the other two Raf isoforms. Several portions of A-Raf were purified throughout this study, but most research concentrated on the conserved region 2 and 3 (CR2 and CR3) domains of A-Raf and the full-length protein. The CR2/CR3 domains and full-length A-Raf were purified by affinity chromatography on a glutathione Sepharose column and column fractions were analyzed by SDS-PAGE. Two different bands measuring approximately 75 kDa and 66 kDa resolved on the SDS-PAGE gel of full-length A-Raf while three bands measuring approximately 75 kDa, 66 kDa and 45 kDa resolved on the SDS-PAGE gel of the CR2/CR3 domains of A-Raf. The CR2/CR3 domains and full-length A-Raf were also extensively studied by mass spectrometry but results were inconclusive. Western blot analysis was also performed on the CR2/CR3 domains and full-length A-Raf. Results indicated that multiple bands were present and that degradation of the protein had taken place. A-Raf was thus deemed unsuitable for crystallization trials and the project was terminated.Mannitol is an acyclic polyalcohol and is used commercially for several purposes including acting as an osmoregulatory compound in several pharmaceuticals and as an artificial sweetener in products targeted for diabetic patients. Commercially, mannitol is produced by the hydrogenation of 50% fructose/50% glucose syrup at high temperatures. However, the product of this process yields an excess of sorbitol and therefore the mannitol requires further purification. Mannitol dehydrogenase catalyzes the conversion of D-fructose to D-mannitol and has therefore been targeted for studies to produce a commercial mannitol bioreactor. The aims of this study included crystallization of the hyperthermophilic Thermotoga maritima (TmMtDH) mannitol dehydrogenase and subsequent X-ray diffraction and structure analysis. Dr. Claire Vieille at Michigan State University provided purified protein for crystallization trials. Two conditions produced diffraction quality crystals of TmMtDH. Condition 1 crystals grew in a solution containing 30% 2-methyl-2,4-pentanediol (MPD) plus 0.1 M HEPES-Na at pH 7.5. Condition 2 crystals grew in a solution containing 15-20% (w/v) polyethylene glycol (PEG) 4000 or 8000 plus 0.1 M sodium citrate at pH 4, 0.2 M sodium bromide and 10% glycerol. Crystals were flash cooled in liquid nitrogen and diffracted on the in-house diffractometer at the Saskatchewan Structural Sciences Center and at beamline 08ID-1 at the Canadian Light Source. Data were collected to 3.3 Å for the crystal that grew in condition 1 but the structure could not be solved before the completion of this project. The space group of the condition 1 crystal was P212121 with unit cell dimensions a = 83.43 Å, b = 120.61 Å, c = 145.76 Å

Material design for OLED lighting applications: Towards a shared computational and photophysical revelation of thermally activated delayed fluorescence

As the third generation of luminescent materials, thermally activated delayed fluorescence (TADF)-type compounds have great potential as emitter molecules in OLEDs allowing for electro-fluorescence with 100 % internal quantum efficiency. For organic electronics, the general wide range of applications from OLEDs, bio-fluorescence imaging to sensor technologies and photonic energy storages roots on the enormous variety of organic materials. Especially in the field of metal- free aromatic designs, the range of possible materials showing diverse triplet harvesting effects is immense, making material development a highly complex task. Firstly, initial efforts in the understanding of the basic concepts behind TADF will be highlighted. A rational design strategy for TADF materials will be illustrated on an innovative material series based on phenylcarbazoles. A reasonable branch of isomers are theoretically constructed and slight stoichiometric modifications are performed to understand how molecular structure and intramolecular steric hindrance affects reverse intersystem crossing (RISC), while simultaneously revealing the strategy for deep blue TADF. The rational design of a bluish green TADF material called 5CzCF3Ph providing CIEy ≤ 0.4 is demonstrated, enabling peak EQE values of 12.1 % with a promising LT50 of 2 hrs at 500 cd∙m-2. Subsequently, the photophysics of five newly designed trimeric donor (D)-acceptor (A)-donor (D) type material compounds, analogue concepts to archetypical TADF designs, highlight the importance of intramolecular electronic couplings between adjacent triplet states for adiabatically-driven TADF, revealing the mechanism of local type triplet state perturbations on 3CT states. The most promising candidate (DMAC-PTO-DMAC) is disclosed and in turn optimized to meet required conditions for deep blue TADF emission. Ultimately, a deep blue luminescent material called isoDMAC-PTO is developed, featuring CIE coordinates of (0.16, 0.14) with an overall quantum yield of (86.4 ± 0.5) %. The focus switches to the fundamental understanding of the underlying mechanism giving rise to TADF in small molecules, leaving the scope of deep blue emission. While investigating the photophysical properties of a synthesized donor (D)-acceptor (A) type thermally activated delayed fluorescence (TADF) emitter named methyl 2-(9,9-dimethylacridin-10-yl)benzoate (DMAC-MB), it is possible to identify the excited state dynamics mediating the spin-flip process and hence the reutilization of non-radiative triplet states allowing for an internal quantum efficiency approaching unity. As experimentally observed by detailed temperature- and time-dependent transient photoluminescence (PL) measurements and consolidated by comprehensive quantum-chemical considerations, excited state configuration interaction by non-adiabatic couplings are anticipated as key property behind triplet up-conversion in the vicinity of conical intersections, contributing to recent research facing the exciton management within the auspicious field of TADF. For the first time, this thesis reports that even a TADF-silent molecule can be converted into efficient TADF systems by increasing the donor π- conjugation length through polymerization of the building block itself. With a total photoluminescence quantum yield up to 71 %, comprehensible research illustrates an efficient thermally activated delayed fluorescence polymer P1, based solely on non-TADF chromophores represented by a model compound 2 (PLQY of 3 % at RT). Finally, as predicted by TDDFT calculations and shown for the first time in the aspiring field of TADF, a thermally activated delayed fluorescence polymer based on a merely radiative, solely phosphorescent repeating unit is demonstrated. Intramolecular π-conjugation is exploited to trigger the charge-transfer excited state energy, revealing a general design tool to provoke TADF, reserved in particular for polymers. While the introduced twisted methyl 2-(9,9-dimethylacridin-10-yl) benzoate (DMAC-MB) reveals efficient thermally activated delayed fluorescence (TADF), a modified analogue 9,9-Dimethyl-5H,9H-quinolino[3,2,1-de]acridin-5-one (DMAC-ACR) shows emerging room temperature phosphorescence (RTP). As for TADF, intramolecular non-adiabatic couplings are unlocked as key feature actuating persistent RTP, linking photophysical analogies between TADF and RTP to structural self-similarities. Last but not least, degradation processes in TADF materials will be addressed. A correlation between theoretically calculated bond-dissociation energies (BDEs) and phenomenological observations reveals that low BDEs, in particular along pronounced charge-transfer bonds, ultimately lead to irreversible TADF material degradation induced by bi-molecular processes comprising TPQ as well as TTA. Finally, this thesis reveals the photophysics of 24 newly designed, synthesized and characterized TADF materials and demonstrates a fundamentally new approach for RTP, based on structural analogues to TADF. Far reaching design principles as conjugation induced TADF in polymers, as well as new design strategies selectively incorporating virbonic couplings yield device performances comprising LT50 of 2 hrs at 500 cd∙m-2 and targeted deep blue emission with CIE (0.16, 0.14). While lighting the way for TADF as future luminescent OLED materials, intrinsic material instabilities due to low bond-dissociation energies are disclosed as key-issues for tomorrows research

Nuclear Magnetic Resonance Spectroscopy

Nuclear Magnetic Resonance (NMR) spectroscopy is a nondestructive technique that can be used to characterize a wide variety of systems. Sustained development of both methodology and instrumentation have allowed NMR to evolve as a powerful technology, with applications in pure sciences, medicine, drug development, and important branches of industry. NMR provides precise structural information down to each atom and bond in a molecule, and is the only method for the determination of structures of molecules in a solution. This book compiles a series of articles describing the application of NMR in a variety of interesting scientific challenges. The articles illustrate the versatility and flexibility of NMR

Targeting farnesyl pyrophosphate synthase of Trypanosoma cruzi by fragment-based lead discovery

Trypanosoma cruzi (T. cruzi) is the causative agent of Chagas disease (CD), which mostly affects underprivileged populations in South and Central America. The current standard of care for this disease are the two empirically discovered drugs benznidazole and nifurtimox. They show low efficacy, difficulties in administration and severe side effects. Moreover, there are T. cruzi strains that have formed resistances. Thus, the development of a safe and efficient drug is urgently needed. T. cruzi is dependent on isoprenoid biosynthesis as ergosterol and other 24 alkylsterols are essential metabolites that cannot be acquired by other mechanisms. Therefore, it was hypothesised that enzymes along this pathway are promising drug targets. A number of compounds targeting these enzymes were tested and have been shown to inhibit parasite growth. Among those enzymes is farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the isoprenoid pathway, which is in the focus of this work. It catalyses the synthesis of farnesyl pyrophosphate (FPP), a C15 building block in sterol biosynthesis and in protein prenylation of signalling proteins. Bisphosphonates (BPs) are known active site directed FPPS inhibitors, which exhibit ideal pharmacokinetics to target bone mineral and are used to treat bone diseases. BPs can also combat T. cruzi flagellates but are not ideal to treat CD due to their pharmacokinetics. In the search for new chemotypes, several non-BP inhibitors that bind to another pocket were found for human FPPS (hFPPS) by fragment based screening (FBS). Recently, it was shown that the product of FPPS, farnesyl pyrophosphate (FPP), can bind to this pocket and locks the enzyme in an open and inactive state, thus showing the allosteric character of this pocket. The current work aims at the discovery of non-BP inhibitors of T. cruzi FPPS (TcFPPS), which could be starting points for the development of a treatment against CD. Towards this goal, recombinant expression in E. coli cells and purification by means of IMAC and SEC yielded pure und homogenous TcFPPS (chapter 5.1). This includes unlabelled, 13C15N labelled and in vivo biotinylated avi-tagged TcFPPS. Furthermore, a novel, reliable, highly reproducible, and well diffracting crystallization system was established. The system exhibits excellent properties for FBS as it was compatible with different types of 96-well plates. Apo crystals were stable for up to 24 h in 15% DMSO and allowed collection of data sets with a diffraction limit of around 1.6 Å. The best achieved diffraction limit was 1.28 Å for a soaked TcFPPS crystal (PDB ID 6R09). The allosteric region in TcFPPS was investigated by means of sequence analysis and structural superimposition of various orthologous FPPSs (chapter 5.2). This revealed that the allosteric region is less conserved than the active site. Differences among residues in equivalent positions that form the allosteric site were observed, which is surprising if it is assumed that all FPPSs can be product inhibited as hFPPS. A remarkable finding is that residue Phe50 in TcFPPS is an exception in an otherwise highly conserved position. It causes steric hindrance of the pocket in TcFPPS. An attempt to reposition established allosteric inhibitors of hFPPS showed binding affinity to TcFPPS but the two obtained crystal structures demonstrated their binding to sites on the protein surface (sites S1 and S2, PDB IDs 6R08 and 6R07, respectively). The Novartis core and fluorine library (1336 and 482 compounds) were screened on TcFPPS, which resulted in 63 and 45 validated fragment hits, respectively (chapter 5.3). Performing the same screen with T. brucei FPPS (TbFPPS), the causative agent of African sleeping sickness, and counter screening on hFPPS led to unique, pairwise and triple binders demonstrating selectivity at the early stage of FBS. Strikingly, TcFPPS has generally more binders than TbFPPS, and TcFPPS has many unique hits when compared to TbFPPS. Subsequent crystallization experiments with the core library hits resulted in 3D structures of two TcFPPS complexes. One ligand binds to the homodimer interface (site S12) and the other one in the active site. The latter was identified by using the statistical analysis tool Pan-Dataset Density Analysis (PanDDA). FBS by X-ray crystallography at the XChem facility in Harwell, UK, and the HTXlab in Grenoble, France, were conducted (chapter 5.4). The XChem screen identified 35 fragment binders (PDB IDs 5QPD – Z, 5QQ0 – 9, 5QQA – C) in binding sites that were distributed over the entire protein. This includes the active site, the allosteric site, the homodimer interface, sites on the surface and a new site in close proximity to the active site. Strikingly, the first two fragments binding to the allosteric site of TcFPPS in its open state were identified. Rotation of the phenyl side chain of Phe50 led to opening of the former closed pocket. The HTXlab screen identified additional binders for the active and allosteric site. In total 1244 data sets were collected and analysed. This process was accelerated using PanDDA. The first fragment-to-lead optimization by means of virtual screening using the web-based platform ANCHOR.QUERY was based on fragment hit LUY (chapter 5.5). Compounds were synthesised using one-pot one-step multi-component reactions. Synthesis of 11 compounds (MCR 1 – 11) was successful, but poor solubility was detrimental in subsequent testing on TcFPPS and crystallization experiments did not lead to a structural model of a complex. A second fragment to lead optimization using a fragment merging approach for chemical optimization was based on the active site directed binders AWM, LVV, LUY, LDV and AWV (chapter 5.6). A library of 12 compounds (MCN 1 – 12) was synthesised by reductive amination. X-ray structures revealed unexpected binding modes for compounds MCN-1, -4 and -8. Instead of retaining the binding site of the fragment, the merged compounds bind to the surface directed binding site S1 (PDB IDs 6R09, 6R0A, 6R0B). Nevertheless, the 50 new crystal structures of TcFPPS fragment complexes discussed in this work will pave the way for future drug discovery campaigns for CD. The large diversity of the fragments’ scaffolds and different binding sites are potential starting points for inhibitors with different physicochemical properties and a novel mode of action that might help to overcome the limitations related to the BP scaffold