11 |
The KIM-family protein-tyrosine phosphatases use distinct reversible oxidation intermediates: Intramolecular or intermolecular disulfide bond formationMachado, Luciana E. S. F., Shen, Tun-Li, Page, Rebecca, Peti, Wolfgang 26 May 2017 (has links)
The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). KIM-PTPs bind and dephosphorylate mitogen-activated protein kinases (MAPKs) and thereby critically modulate cell proliferation and differentiation. PTP activity can readily be diminished by reactive oxygen species (ROS), e.g. H2O2, which oxidize the catalytically indispensable active-site cysteine. This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). Critically, our understanding of ROS-mediated PTP oxidation is not yet sufficient to predict the molecular responses of PTPs to oxidative stress. However, identifying distinct responses will enable novel routes for PTP-selective drug design, important for managing diseases such as cancer and Alzheimer's disease. Therefore, we performed a detailed biochemical and molecular study of all KIM-PTP family members to determine their H2O2 oxidation profiles and identify their reversible inactivation mechanism(s). We show that despite having nearly identical 3D structures and sequences, each KIM-PTP family member has a unique oxidation profile. Furthermore, we also show that whereas STEP and PTPRR stabilize their reversibly oxidized state by forming an intramolecular disulfide bond, HePTP uses an unexpected mechanism, namely, formation of a reversible intermolecular disulfide bond. In summary, despite being closely related, KIM-PTPs significantly differ in oxidation profiles. These findings highlight that oxidation protection is critical when analyzing PTPs, for example, in drug screening.
|
12 |
Investigation of protein-protein interactions involving retinoblastoma binding protein 6 using immunoprecipitation and nuclear magnetic resonance spectroscopyChen, Po-An January 2019 (has links)
>Magister Scientiae - MSc / Retinoblastoma Binding Protein 6 (RBBP6) is a 200 KDa multi-domain protein that has been
shown to play a role in mRNA processing, cell cycle arrest and apoptosis. RBBP6 interacts with
tumour suppressor proteins such as p53 and pRb and has been shown cooperate with Murine
Double Minute 2 (MDM2) protein in catalyzing ubiquitination and suppression of p53.
Unpublished data from our laboratory has suggested that RBBP6 and MDM2 interact with each
other through their RING finger domains. RBBP6 has also been shown to have its own E3 ubiquitin
ligase activity, catalyzing ubiquitination of Y-Box Binding Protein 1 (YB-1) in vitro and in vivo. YB-
1 is a multifunctional oncogenic protein that is generally associated with poor prognosis in cancer,
tumourigenesis, metastasis and chemotherapeutic resistance. Unpublished data from our
laboratory shows that RBBP6 catalyzes poly-ubiquitination of YB-1, using Ubiquitin-conjugating
enzyme H1 (UbcH1) as E2 ubiquitin conjugating enzyme. / 2022-02-25
|
13 |
Anvil cell gasket design for high pressure nuclear magnetic resonance experiments beyond 30 GPaMeier, Thomas, Haase, Jürgen 28 May 2018 (has links)
Nuclear magnetic resonance (NMR) experiments are reported at up to 30.5 GPa of pressure using radiofrequency (RF) micro-coils with anvil cell designs. These are the highest pressures ever reported with NMR, and are made possible through an improved gasket design based on nano-crystalline powders embedded in epoxy resin. Cubic boron-nitride (c-BN), corundum (α-Al2O3), or diamond based composites have been tested, also in NMR experiments. These composite gaskets lose about 1/2 of their initial height up to 30.5 GPa, allowing for larger sample quantities and preventing damages to the RF micro-coils compared to precipitation hardened CuBe gaskets. It is shown that NMR shift and resolution are less affected by the composite gaskets as compared to the more magnetic CuBe. The sensitivity can be as high as at normal pressure. The new, inexpensive, and simple to engineer gaskets are thus superior for NMR experiments at high pressures.
|
14 |
Moissanite anvil cell design for giga-pascal nuclear magnetic resonanceMeier, Thomas, Herzig, Tobias, Haase, Jürgen 28 May 2018 (has links)
A new design of a non-magnetic high-pressure anvil cell for nuclear magnetic resonance (NMR) experiments at Giga-Pascal pressures is presented, which uses a micro-coil inside the pressurized region for high-sensitivity NMR. The comparably small cell has a length of 22 mm and a diameter of 18 mm, so it can be used with most NMR magnets. The performance of the cell is demonstrated with externalforce vs. internal-pressure experiments, and the cell is shown to perform well at pressures up to 23.5 GPa using 800 μm 6H-SiC large cone Boehler-type anvils. 1H, 23Na, 27Al, 69Ga, and 71Ga NMR test measurements are presented, which show a resolution of better than 4.5 ppm, and an almost maximum possible signal-to-noise ratio.
|
15 |
Content determination of explosive precursors and narcotic salts using 35Cl-nuclear magnetic resonanceBergqvist, Sandra January 2023 (has links)
Explosive precursors and narcotic salts are chemicals contributing to an undesirabledevelopment of the Swedish society, both in terms of criminal activities and harm to the environment. Reducing the illegal use of these chemicals is important in the work towards a safer society. National Forensic Centre (NFC) is the state agency responsible for forensic investigations for the Swedish Police Authority. The Drug Analysis and ChemistryTechnology section at NFC were both in need for an accurate quantification method to determine the content of Cl in narcotic salts and explosive precursors. Nuclear magnetic resonance (NMR) spectroscopy was assessed to be suitable since a recently published article had shown applicability of 35Cl NMR on narcotic salts. The aim of the method was to find the most appropriate parameter settings for the compounds of interest, including operating frequency, 90° pulse length, number of scans, relaxation time, and relaxation delay. To ensure a reliable and accurate method, the following validation parameters were studied; linearity, limit of detection (LOD), limit of quantification (LOQ), intermediate precision, trueness, repeatability, and ruggedness. Dimethyl sulfoxide (d6-DMSO) was chosen as the preferredsolvent for the Drug Analysis section since it is a common solvent for their 1H-NMR analysis. For explosive precursors results showed advantages of using deuterium oxide (D!O) as solvent, considering accuracy, solubility and shorter analysis time.Concluding, the chosen criteria of signal-to-noise (S/N) ratio >6 resulted in an LOQ of around 0.15g/L, though this was dependent upon the number of scans utilized. Successful pulse length experiments determined exact 90° pulse lengths for each sample and solvent combination. The longitudinal relaxation time T1 was also successfully determined, and since it was multiplied with five to ensure complete relaxation to stable state the relaxation delay D1was assumed as an insignificant parameter for the determination of chloride. Quantification was based upon the pulse-length based concentration determination (PULCON) using an external standard. The ruggedness can be studied additionally by another experienced operator (since trueness was strongly dependent upon the preparation of the external standard solution). The method displayed good linearity over the mass range normally utilized in such quantifications. The conclusion drawn in the thesis is that the method shows great promise but additional analyzes are still required before implementation at NFC
|
16 |
Cytotoxic Alkaloids from Microcos paniculata with Activity at Neuronal Nicotinic ReceptorsStill, Patrick C. 09 August 2013 (has links)
No description available.
|
17 |
Making wood durable. A sustainable approachwith linseed oil / Att göra trä beständigt. Ett hållbart tillvägagångssätt med linoljaOlsson, Helena January 2019 (has links)
Linseed oil has been and is used for vast number of applications, such as in food and paint industry, and wood preservation. It is a good environmental choice, as it originates from renewable sources. Linseed oil is mainly a mixture of triglyceride of fatty acids, both saturated and mono- or polyunsaturated, which allows the oil to oxidize. The oxidation occurs via an auto-oxidation mechanism with the carbon-carbon double bonds and oxygen from the air, reacts to form a polymer. Herein, four different linseed oils (three commercial ones and one industrially available) were analyzed to obtain a better understanding of why different oils provide different protection of wooden materials. This was done by a study of the unoxidized oil, followed by an oxidation time-resolved study of oxidized oil films. The analysis was done by nuclear magnetic resonance, gas chromatography - mass spectrometry and/or inductively coupled plasma atomic emission spectroscopy. This study provided the fatty acid profile of the oils, which were similar for all oils. The unoxidized oils contained some metals ions which probably originate from additives. Aluminum, cobalt, iron, manganese, and zinc was detected in some of the oils at concentrations up to 135 mg/L, but only manganese was detected in all oils and its concentration was much higher than all other metals together. The time-resolved oxidation study had some problems with the solubility of the formed polymers. Several solvents were examined, such as dimethylsulfoxide, alkaline alcohol solutions and toluene, before chloroform-d was chosen as solvent. Though, chloroform-d was not a perfect solvent; it was capable to solve a fraction of the sample, but the fraction decreased with oxidation time. After fifteen days of oxidation, only a few percent of the sample could be dissolved, but for short oxidation times (<48 h) the majority of the samples were dissolved. The oils were analyzed after thirteen different oxidation times. Some structural changes appeared, for example loss of unsaturated protons and some oxidation products arose, such as peroxides and aldyhydes. The diffusion coefficient decreased over the first 3-4 days of oxidation, as expected when the polymerization progressed. After a week of oxidation, the diffusion coefficient increased again, this could possibly be explained by the solubility problem for the large polymer formed. Contrary, at shorter oxidation times this method probably could still be used, as the majority of the sample was dissolved. However, the solubility problem made it impossible to conclude anything about the oxidation rate at longer oxidation times and thus prevented any ranking of the oils. / Linolja har använts och används för många olika tillämpningar, till exempel i mat- och färgindustrin, samt för att bevara trä. Det är klimatmässigt ett bra val, då det kommer från en förnyelsebar källa. Linolja innehåller huvudsakligen en blandning av triglycerider av fettsyror, som både kan vara mättade, enkelomättade eller fleromättade, detta gör att linolja kan oxidera och torka. Oxidationen sker via en auto-oxidation mekanism, med kol-kol dubbelbindningarna och syre från luften som producerar till en polymer. I detta projekt undersöktes fyra olika linoljor (tre kommersiella och en industriell), för att ge en bättre förståelse till varför olika oljor ger olika bra skydd för trämaterial. Detta gjordes genom att undersöka de icke-oxiderade oljorna och sedan göra en tidsstudie på oxiderade oljefilmer. Alla dessa prover analyserades med nuclear magnetic resonance, gas chromatography - mass spectrometry and/or inductively coupled plasma atomic emission spectroscopy. Studien gav resultat på sammansättningen av fettsyror i oljorna, vilken var liknande för alla oljorna. De icke-oxiderade oljorna innehöll ett par metaller, som förmodligen kommer från additiv. Aluminium, kobolt, järn, mangan och zink hittades i några av oljorna i koncentrationer upp till 135 mg/L, men bara mangan var detekterad i alla oljorna och dess koncentration var högre än alla andra metaller tillsammans. Tidsstudien hade problem med lösligheten av proverna. Flertalet lösningsmedel undersöktes, exempelvis dimetylsulfoxid, alkaliska alkohollösningar och toluen, innan kloroform-d valdes som lösningsmedel. Däremot var kloroform-d inte ett perfekt lösningsmedel, den hade förmågan att lösa en del av proverna, men den delen minskade med oxidationstid. Efter femton dagar kunde den bara lösa ett par procent, men efter kortare oxideringstider (<48 h) gick majoriteten av proven att lösa. Oljorna analyserades vid tretton olika oxideringstider. Några strukturella förändringar uppmättes, till exempel minskade mängden dubbelbindningar, och ett par biprodukter från oxideringen detekterades, så som peroxider och aldehyder. Diffusionskonstanterna för oljorna minskade under de första 3-4 dagarna av oxidering, precis som förväntat under polymeriseringen. Efter en vecka av oxidering ökade diffusionskonstanterna igen, det kan förmodligen förklaras av löslighetsproblemen, då diffusionskonstanten är beroende av koncentrationen. Å andra sidan, vid kortare oxideringstider kan denna metod fortfarande användas, eftersom vid denna tidpunkt löste sig fortfarande majoriteten av provet. Dock, löslighetsproblemet gjorde det omöjligt att dra slutsatser kring längre oxideringstider och därmed förhindrades rangordning av oljorna.
|
18 |
Structural characterisation of novel poly-aryl compoundsKhashoqji, Moayad January 2016 (has links)
Poly-aryl, also known as polyphenylene compounds are a class of dendrimer, which contain a large number of aromatic rings. They are of interest because they display restricted rotation of their stearically congested aromatic rings. These extended structures have the potential to act as precursors for even larger aromatic systems and have many applications including electronic devices, drug delivery and catalysis. A total of 23 novel poly-aryl compounds have been examined using single crystal X-ray diffraction and a number of structural patterns have emerged. Six of the compounds contain alkynes and it has been observed that their conformation is governed by a combination of conjugation between the alkyne and aryl groups and inter-molecular interactions. In the more extended poly-aryl compounds steric congestion rules out any possibility of conjugation between the rings and their conformation is governed by intra-molecular non-bonded interactions in the core of the molecules and by inter-molecular interactions in their periphery. Where possible, solution NMR measurements were carried out on the poly-aryl compounds and confirmed that the solution structures are in agreement with those obtained from individual crystal.
|
19 |
Quantum Algorithms Using Nuclear Magnetic Resonance Quantum Information ProcessorMitra, Avik 10 1900 (has links)
The present work, briefly described below, consists of implementation of several quantum algorithms in an NMR Quantum Information Processor.
Game theory gives us mathematical tools to analyze situations of conflict between two or more players who take decisions that influence their welfare. Classical game theory has been applied to various fields such as market strategy, communication theory, biological processes, foreign policies. It is interesting to study the behaviour of the games when the players share certain quantum correlations such as entanglement. Various games have been studied under the quantum regime with the hope of obtaining some insight into designing new quantum algorithms. Chapter 2 presents the NMR implementation of three such algorithms. Experimental NMR implementation given in this chapter are:
(i) Three qubit ‘Dilemma’ game with corrupt sources’. The Dilemma game deals with the situation where three players have to choose between going/not going to a bar with a seating capacity of two. It is seen that in the players have a higher payoff if they share quantum correlations. However, the pay-off falls rapidly with increasing corruption in the source qubits. Here we report the experimental NMR implementation of the quantum version of the Dilemma game with and without corruption in the source qubits.
(ii) Two qubit ‘Ulam’s game’. This is a two player game where one player has to find out the binary number thought by the other player. This problem can be solved with one query if quantum resources are used. This game has been implemented in a two qubit system in an NMR quantum information processor.
(iii) Two qubit ‘Battle of Sexes’ game. This game deal with a situation where two players have conflicting choices but a deep desire to be together. This leads to a dilemma in the classical case. Quantum mechanically this dilemma is resolved and a unique solution emerges. The NMR implementation of the quantum version of this game is also given in this chapter.
Quantum adiabatic algorithm is a method of solving computational problems by evolving the ground state of a slowly varying Hamiltonian. The technique uses evolution of the ground state of a slowly varying Hamiltonian to reach the required output state. In some cases, such as the adiabatic versions of Grover’s search algorithm and Deutsch-Jozsa algorithm, applying the global adiabatic evolution yields a complexity similar to their classical algorithms. However, if one uses local adiabatic evolutions, their complexity is of the order √N (where N=2n) [37, 38]. In Chapter 3, the NMR implementation of (i) the Deutsch-Jozsa and the (ii) Grover’s search algorithm using local adiabatic evolution has been presented. In adiabatic algorithm, the system is first prepared in the equal superposition of all the possible states which is the ground state of the beginning Hamiltonian. The solution is encoded in the ground state of the final Hamiltonian. The system is evolved under a linear combination of the beginning and the final Hamiltonian. During each step of the evolution the interpolating Hamiltonian slowly changes from the beginning to the final Hamiltonian, thus evolving the ground state of the beginning Hamiltonian towards the ground state of the final Hamiltonian. At the end of the evolution the system is in the ground state of the final Hamiltonian which is the solution. The final Hamiltonian, for each of the two cases of adiabatic algorithm described in this chapter, are constructed depending on the problem definition.
Adiabatic algorithms have been proved to be equivalent to standard quantum algorithms with respect to complexity [39]. NMR implementation of adiabatic algorithms in homonuclear spin systems face problems due to decoherence and complicated pulse sequences. The decoherence destroys the answer as it causes the final state to evolve to a mixed state and in homonuclear systems there is a substantial evolution under the internal Hamiltonian during the application of the soft pulses which prevents the initial state to converge to the solution state. The resolution of these issues are necessary before one can proceed for the implementation of an adiabatic algorithm in a large system. Chapter 4 demonstrates that by using ‘strongly modulated pulses’ for creation of interpolating Hamiltonian, one can circumvent both the problems and thus successfully implement the adiabatic SAT algorithm in a homonuclear three qubit system. The ‘strongly modulated pulses’ (SMP) are computer optimized pulses in which the evolution under the internal Hamiltonian of the system and RF inhomogeneities associated with the probe is incorporated while generating the SMPs. This results in precise implementation of unitary operators by these pulses. This work also demonstrates that the strongly modulated pulses tremendously reduce the time taken for the implementation of the algorithm, can overcome problems associated with decoherence and will be the modality in future implementation of quantum information processing by NMR.
Quantum search algorithm, involving a large number of qubits, is highly sensitive to errors in the physical implementation of the unitary operators. This can put an upper limit to the size of the data base that can be practically searched. The lack of robustness of the quantum search algorithm for a large number of qubits, arises from the fact that stringent ‘phase-matching’ conditions are imposed on the algorithm. To overcome this problem, a modified operator for the search algorithm has been suggested by Tulsi [40]. He has theoretically shown that even when there are errors in implementation of the unitary operators, the search algorithm with his modified operator converges to the target state while the original Grover’s algorithm fails. Chapter 5, presents the experimental NMR implementation of the modified search algorithm with errors and its comparison with the original Grover’s search algorithm. We experimentally validate the theoretical predictions made by Tulsi that the introduction of compensatory Walsh-Hadamard and phase-flip operations refocuses the errors.
Experimental Quantum Information Processing is in a nascent stage and it would be too early to predict its future. The excitement on this topic is still very prevalent and many options are being explored to enhance the hardware and software know-how. This thesis endeavors in this direction and probes the experimental feasibility of the quantum algorithms in an NMR quantum information processor.
|
20 |
Solution NMR Studies Of E.Coli Acetohydroxy Acid Synthase (AHAS) IMitra, Ashima 03 1900 (has links)
Branched chain amino acids are classified as essential amino acids since their biosynthetic routes or pathways are restricted only to micro-organisms, fungi and plants. Given their unique distribution, the enzymes of the branched chain amino acid biosynthetic pathway are ideal targets for the development of herbicides, anti-bacterials and potentially antifungal agents.
Acetohydroxy acid synthase (AHAS) catalyses the firs step in the biosynthesis of branched chain amino acids. AHAS activity had been first identified in extracts of E. coli as early as in 1958 by Brown and Umbarger . Ever since its discovery, AHAS have been found to exist in all eubacteria, archaebacteria, algae, yeast and plants. The enzymatic properties of prokaryotic and eukaryotic AHASs have been thoroughly investigated. A single isoform of the enzyme is known to exist in all organisms except in enterobacteria which have three isoforms of the enzyme. Activity of the three isoforms of E. coli AHAS (I, II and III) have been studied using various biochemical and biophysical methods. AHAS enzyme expressed in bacteria and yeast are heterotetrameric composed two large catalytic and two small regulatory subunits. While much has been learnt from the structure of the catalytic subunits (yeast and Arabidopsis thaliana) and the regulatory subunits (regulatory subunit of E. coli AHAS III) in isolation, the structural properties of the holoenzyme remain unexplored.
AHAS is unique from the point of view that it exhibits a striking domain organization in the catalytic subunit and also in the regulatory subunits. Thus understanding the nature of protein – protein interactions both as domain – domain interactions within the subunit as well as protein – protein interactions across subunits is crucial to understanding the structural basis for the activity and regulation of this important enzyme. Of these, understanding the structural basis for the interaction between the regulatory and the catalytic subunits within the holoenzyme is paramount. The poor solubility and the intrinsic instability of the proteins have hampered the efforts to structurally characterize any of the AHAS holoenzymes. An active AHAS I construct has been created by Vyazmensky et. al., where the catalytic and the regulatory subunit have been expressed together as a single chain separated by a flexible linker. While this single chain construct is catalytically active, there have been no reports of successful crystallization of this single chain AHAS I enzyme.
The crystallographically determined structure of the catalytic subunit of yeast and A. thaliana AHAS has shown that the protein is composed of three independently folded domains, α, β and γ. More importantly the polypeptide sequence of the catalytic subunits of AHAS across all species is largely conserved. This indicates that the overall tertiary folds of the catalytic subunit would be alike. The unique domain architecture of the AHAS catalytic subunit and the relatively small size of the regulatory subunit forms the basis for implementation of a novel strategy, in which structural interactions between the domains (catalytic site as well as the non catalytic site interactions) as well as structural interactions between the domains of the catalytic and the regulatory subunit of E. coli AHAS I can be explored in an incremental manner. Initiation of structural characterization of the individual domains of the catalytic subunit of E. coli AHAS I and understanding the structural basis of the interaction between the domains of the catalytic and the regulatory subunits of the protein, using solution NMR methods, forms the theme for this study.
The domains of the catalytic subunit (ilvB) of E.coli AHAS I were identified based on the similarity in the sequence of this subunit with the yeast protein and the structural information of the yeast protein. The individual domains of the ilvB protein (ilvBα, ilvBβ and ilvBγ) and ilvN, the regulatory subunit of AHAS I, were cloned, expressed and purified for structural studies. The problem of poor expression and solubility profiles of the AHAS proteins was circumvented with the help of a novel cytb5 fusion system developed in our laboratory during the course of this study. The high expression levels of the fusion protein in minimal medium enabled the preparation of isotopically (15N, 13C/15N, 2H/13C/15N) enriched samples of the proteins in a cost effective manner. The cytb5 fusion system has provided very uniform and reliable expression of these proteins without accumulation of any protein in the insoluble fraction.
From the structure of the catalytic subunit of yeast AHAS it is known that the α and γ domains of the protein interact to form the active site. The two domains provide group specific interaction sites for anchoring the co-factor TPP in an appropriate conformation for catalysis. The β domain on the other hand does not directly participate in the ormation of the active site but anchors the co-factor FAD which in turn plays a structural role in enzyme catalysis. In the present study we employed biochemical and biophysical methods to establish the structural integrity of the individually expressed domains of the catalytic subunit (ilvB) and the regulatory subunit of AHAS I. Reactions catalyzed by enzymes formed by assembling different domain and subunits indicate that the proteins when reconstituted in vitro form a catalytically competent complex. Formation of S-acetolactate, the product of the reaction catalyzed by the AHAS I holoenzyme, has been confirmed using colorimetric as well as spectroscopic methods such as CD and NMR.
Multinuclear, multidimensional NMR methods have been utilized to obtain sequence specific assignments of apo - ilvBβ (non FAD bound form). Preparation of an NMR amenable sample of ilvBβ proved to be the rate limiting step due to the predisposition of the protein to undergo aggregation at concentrations required for solution NMR studies. However, careful screening of large number of buffer conditions enabled us to establish an optimum sample condition where the protein was soluble, stable and free of aggregation and hence suitable for NMR studies. Uniformly enriched 15N, 13C/15N, and 2H/13C/15N samples of ilvBβ were prepared to obtain sequence specific assignments and secondary structural information. From the secondary chemical shifts of backbone 13Cα atoms and short and medium range NOEs the secondary structure of the non FAD bound (apo) form of ilvBβ has been determined. Using chemical shift mapping methods, the residues of the ilvBβ domain that are involved in FAD binding have been identified. The distribution of the secondary structural elements and the residues that are involved in binding the co-factor FAD were found to be conserved for the E. coli and yeast proteins. This suggests that the tertiary Fold of the FAD binding β domain of the catalytic subunit of E. coli AHAS is identical to that in the yeast protein.
The interaction between the individual domains of ilvB and ilvN (the regulatory subunit) has been investigated using spectroscopic methods. Changes in CD spectra indicate that ilvN interacts with ilvBα and ilvBβ domains of the catalytic subunit and not with the ilvBγ domain. NMR chemical shift mapping methods has shown that ilvN binds close to the FAD binding site in ilvBβ and proximal to the intra-subunit ilvBα/ilvBβ domain interface. The implication of this interaction and the role of the regulatory subunit on the activity of the holoenzyme are discussed.
|
Page generated in 0.0606 seconds