Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex: e1 and e2 substrate specificty, e1 carboligase activity, and e2 interchain succinyl transfer

Escherichia coli (E. coli) 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc) contains three components: a thiamin di phosphate (ThD P) dependent 2-oxogl utarate dehydrogenase (E1 o), a di hydrol i poylsucci nyl transferase (E2o), and a di hydrol i poyl dehydrogenase (E3). The first two components carry out the principal reactions for succinyl CoA formation while the third one reoxidizes dihydrolipoamide to lipoamide. This mechanism is similar to other 2-oxoacid dehydrogenase complexes, including pyruvate dehydrogenases (PDHc) and branched-chain dehydrogenases. E1o of the E. coli OGDHc was engineered to accept unnatural substrates. The natural substrate for E1 o is 2-oxogl utarate (2-OG) and the enzyme was engineered to accept substrates lacking the 5-carboxylate group, 2-oxovalerate (2-OV). E1o was subjected to saturation mutagenesis at H is260 and H is298. The H is298Asp E1 o variant was identified in the screen to accept the unnatural substrate. In addition, it was discovered that His260 is required for substrate recognition, but His298 could be replaced with hydrophobic residues of similar molecular volume. To interrogate whether the second component would allow synthesis of acyl-coenzyme A derivatives, hybrid complexes consisting of recombinant components of OGDHc (o) and pyruvate dehydrogenase (p) enzymes were constructed, suggesting that a different component is the “gatekeeper” for specificity for these two multienzyme complexes in bacteria, E1 p for pyruvate but E2o for 2-OG. Although His298Asp E1o accepted 2-OV, reconstitution of the variant with E2o and E3 did not generate NADH in the overall reaction using 2-OV. Hence, the reaction may be hindered in the E2o component. E2o consists of an amino-terminal lipoyl domain (E2oLD, 12 kDa), followed by a peripheral subunit binding domain (4 kDa) and a succinyltransferase domain (E2oCD, 28 kDa). There are two possibilities for the failure to form NADH. Reductive acylation is not occurring in the E2oLD or acyl transfer to CoA is not taking place in E2oCD. His298Asp E1o, E2oLD, and 2-OV form butyrylated E2oLD, which was shown by electrospray ionization mass spectrometry. Therefore, the E2oCD domain necessitates optimization to produce acyl-CoA derivatives. Succinyl transfer to the CD domain may occur through an intrachain or interchain pathway. The E2oLD and E2o with a lysine to alanine substitution at position 43 (E2oK43A) were created by site directed mutagenesis. It is clearly shown that E2oLD was capable of rescuing the crippled E2oK43A variant by measuring the NADH production in the overall reaction. Therefore, an interchain mechanism is likely between two different E2o subunits. ThDP-dependent enzymes have the potential to be used for chemical synthesis. These enzymes share a common feature in that they catalyze carboligase reactions. E1o catalyzed carboligation products with a variety of substrates and acceptors that vary in the size and functional groups. Structures of the products were confirmed with NMR. In addition, high enantiomeric excess (ee) values were found for the products as shown by chiral gas chromatography and CD spectroscopy. Finally, it was shown that E1o is capable of forming stable esters. This is important because when the carboligase reactions produce 0-ketoacids, these products are unstable and prone to decarboxylation

3D flash memory cube design utilizing COTS for space

With the rapid growth in scope for space missions, the computing capabilities of on-board spacecraft is becoming a major limiting factor for future missions. This thesis is part of a project that designs a radiation hardened NAND Flash memory cube for use in space. The main focus of this thesis is on presenting a new NAND Flash memory cube design with features to allow the cube to be used in space. Another goal of the thesis is to show proof of concept of the cube. Thus, a preliminary RTL was designed for the memory controllerportion of the design and simulated results are shown in the later part of the thesis.M.S

Controlling competing interactions at oxide interfaces: Enhanced anisotropy in La0.7Sr0.3MnO3 films via interface engineering

We investigated thin La0.7Sr0.3MnO3-SrTiO3 heterostructures, where the band alignment is engineered by a variation of La/Sr stoichiometry only at the interface. In thin films, the engineered interface leads to an enhancement of the reversed spin configuration that mimics bulk behavior. Microscopically, this enhancement is closely connected with an increased magnetic anisotropy as well as intercoupling between an e(g) orbital reconstruction and a corresponding anisotropic lattice fluctuation. Furthermore, a reentrant-type behavior, triggered by this intercoupling, is observed in the remanent spin state. This microscopic perspective leads to insights on developing new strategies for maintaining bulk-like properties even in very thin La0.7Sr0.3MnO3 heterostructures.open11910Ysciescopu

Growth model with restricted surface relaxation

We simulate a growth model with restricted surface relaxation process in d=1 and d=2, where d is the dimensionality of a flat substrate. In this model, each particle can relax on the surface to a local minimum, as the Edwards-Wilkinson linear model, but only within a distance s. If the local minimum is out from this distance, the particle evaporates through a refuse mechanism similar to the Kim-Kosterlitz nonlinear model. In d=1, the growth exponent beta, measured from the temporal behavior of roughness, indicates that in the coarse-grained limit, the linear term of the Kardar-Parisi-Zhang equation dominates in short times (low-roughness) and, in asymptotic times, the nonlinear term prevails. The crossover between linear and nonlinear behaviors occurs in a characteristic time t_c which only depends on the magnitude of the parameter s, related to the nonlinear term. In d=2, we find indications of a similar crossover, that is, logarithmic temporal behavior of roughness in short times and power law behavior in asymptotic times

Architecture of the chromatin remodeler RSC and insights into its nucleosome engagement.

Eukaryotic DNA is packaged into nucleosome arrays, which are repositioned by chromatin remodeling complexes to control DNA accessibility. The Saccharomyces cerevisiae RSC (Remodeling the Structure of Chromatin) complex, a member of the SWI/SNF chromatin remodeler family, plays critical roles in genome maintenance, transcription, and DNA repair. Here, we report cryo-electron microscopy (cryo-EM) and crosslinking mass spectrometry (CLMS) studies of yeast RSC complex and show that RSC is composed of a rigid tripartite core and two flexible lobes. The core structure is scaffolded by an asymmetric Rsc8 dimer and built with the evolutionarily conserved subunits Sfh1, Rsc6, Rsc9 and Sth1. The flexible ATPase lobe, composed of helicase subunit Sth1, Arp7, Arp9 and Rtt102, is anchored to this core by the N-terminus of Sth1. Our cryo-EM analysis of RSC bound to a nucleosome core particle shows that in addition to the expected nucleosome-Sth1 interactions, RSC engages histones and nucleosomal DNA through one arm of the core structure, composed of the Rsc8 SWIRM domains, Sfh1 and Npl6. Our findings provide structural insights into the conserved assembly process for all members of the SWI/SNF family of remodelers, and illustrate how RSC selects, engages, and remodels nucleosomes

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging. Photoconvertible proteins occupy two color channels thereby limiting multicolour localisation microscopy applications. Here the authors present UnaG, a new green-to-dark photoswitching fluorescent protein for super-resolution imaging, whose activation is based on a noncovalent binding with bilirubin

Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability