Application of permeabilized yeast cells for biocatalysis

The phosphoenolpyruvate: sugar phosphotransferase system (PTS) is responsible for the uptake and concomitant phosphorylation of many sugars in several species of bacteria. The first step of the PTS involves the phosphorylation of histidine containing phosphocarrier protein (HPr) by enzyme I (E.C. 2.7.3.9), with phosphoenolpyruvate (PEP) serving as the phosphoryl donor. Enzyme I has logically been viewed as a potential target for regulation of the PTS. This thesis presents important information regarding the structure and function of enzyme I of <i>Escherichia coli</i> and <i>Salmonella typhimurium</i>. Fluorescence polarization analysis, although incomplete, showed that the interaction of HPr with enzyme I and with enzyme IIA glc are of low affinity, with a Kd of roughly 10-100 [mu]M. An enzyme I binding site on HPr was determined by a kinetic assay, using site-directed mutants of HPr as substrates for enzyme I. This site of interaction agreed very well with that found in the NMR solution structure of the complex of HPr with the N-terminal domain of enzyme I (Garrett 'et al'., 1999), with a few important differences. Genes encoding mutant enzymes I were cloned from 'S. typhimurium ' strains and the purified proteins were analyzed. The Arg126Cys mutant was defective in phosphotransfer, while the Gly356Ser and Arg375Cys mutants were defective in dimerization and PEP-binding. Intragenic complementation was observed between purified Arg126Cys and Gly356Ser or Arg375Cys enzymes I, through formation of heterodimers. This heterodimers were unstable, and stability depended upon dilution of the enzyme and PEP concentrations. Other site-directed mutants of ,<i>E. coli</i> enzyme I were created, which indicated the importance of the residues Asn352 and Leu355 in dimerization, and Arg296 in PEP-binding. These data led to the conclusions that dimerization and PEP-binding of enzyme I are closely linked cooperative events, and that the monomer:dimer equilibrium of enzyme I, with the dimer being the most active form, may have significant physiological importance. Regulation of the monomer:dimer equilibrium may provide a key target for modulation of the activity of enzyme I, and thus regulation of the PTS.

<p>The natural progression from the introduction of an antibiotic into the market to the emergence of resistant strains demands the constant influx of new drugs to treat infection. Ramoplanin A2, a drug with demonstrated resistance against antibiotic-resistant Gram-positive pathogens, is currently in clinical trials. Ramoplanin is composed of seventeen amino acids condensed with a N-acylated fatty acid tail and a sugar moiety. The biosynthesis of this important metabolite is performed by a host of enzymes including non-ribosomal peptides synthetases, fatty acid biosynthetic enzymes, glycosyltransferases, and other tailoring enzymes. </p><p> In the introductory chapter, the significance and mechanism of the family of ramoplanin antibiotics will be discussed. The biosynthetic cluster of the lipoglycodepsipeptide ramoplanin and its sister antibiotic enduracidin will be outlined and the previous work on this cluster summarized. The proposed formation of the critical N-acylated tail and the parallels to fatty acid biosynthesis will also be described in detail. Chapter two describes the development of an expression system to successfully isolate proteins of interest from the ramoplanin biosynthetic cluster. In addition to increasing the yields of proteins already expressing in heterologous hosts, this technique facilitated the isolation of previously unattainable proteins. This chapter also details the demonstration of their activity through a variety of biochemical techniques. </p><p> Chapter three details the kinetic characterization of Ramo16, a NAD-dependent &#946;-ketoacyl reductase, critical for the formation of the N-acylated fatty acid chain attached to Ramoplanin. Sequence analysis and characterization of the product of the Ramo16 reaction also implies that the biosynthesis of the N-acylated tail of ramoplanin bears significant similarity to type II bacterial fatty acid biosynthesis. These observations have led to a new proposal for the biosynthesis of the N-acylated fatty acid attached to ramoplanin. Further supporting these conclusions, chapter four details the examination of the stereochemistry of the hydrogen removed from the nicotinamide adenine dinucleotide cofactor and the stereochemistry of the product of the enzymatic reaction.</p><p> Chapter five describes the 1.4 Å crystal structure of ramoplanin in the presence of CTAB, an amphipathic membrane phospholipid mimetic. Observation of the location of the N-acylated fatty acid allows interpretation of its critical role in the activity of ramoplanin. The formation of the dimer and the interactions between the two molecules of ramoplanin and ramoplanin and the surroundings were examined in detail and supported earlier structure activity relationships. These observations have led to a new model for ramoplanin and Lipid II recognition in the context of the bacterial membrane. Enduracidin, ramoplanin's sister antibiotic, is produced by S. fungicidicus. Development of fermentation conditions for this strain allowed the isolation of appreciable amounts of enduracidin and 15N-labeled enduracidin, which was used to examine enduracidin's binding to Park's nucleotide, a cell wall precursor to Lipid II, by 2D 1H- 15N HSQC NMR. Although chemical shifts in the HSQC spectrum indicated binding, insoluble fibril formation complicated interpretation and necessitated the use of crystallography to determine the interaction with the substrate. Initial crystal conditions have been determined and diffraction images have been collected at 3.0 Å. Higher resolution images are pending to determine this structure.</p><p> Finally, chapter six discusses the development of a technique to transform S. fungicidicus and introduce plasmids to obtain deletion and insertional disruption mutants. A high throughput PCR screen was determined to enable large batch screening of potential mutants. This technique may be applied to study the function of genes in the pathway by fermenting production of enduracidin and examining the results.</p> / Dissertation

<p>Interleukin 7 receptor, IL7R, is expressed exclusively on cells of the lymphoid lineage and its expression is crucial for development and maintenance of T cells. While transcriptional regulation of IL7R expression has been widely studied, its posttranscriptional regulation has only recently been uncovered. Alternative splicing of IL7R exon 6, the only exon that encodes the transmembrane domain of the receptor, results in membrane-bound (exon 6 included) and soluble (exon 6 skipped) IL7R isoforms, respectively. Interestingly, the inclusion of exon 6 is affected by a single-nucleotide polymorphism associated with the risk of developing multiple sclerosis, a prototypic demyelinating disease of the central nervous system. Given the potential association of exon 6 inclusion with multiple sclerosis, we investigated the cis-acting elements and trans-acting factors that regulate exon 6 splicing.</p><p>We utilized mutagenesis of exon 6 and surrounding introns to identify multiple exonic and intronic cis-acting regulatory elements that impact inclusion of exon 6. At least two of these elements, one exonic splicing silencer and one exonic splicing enhancer, are located in the direct vicinity of the MS-associated SNP. We also uncovered a consensus polyadenylation signal, AAUAAA in intron 6 of IL7R, 16 nucleotides downstream from exon 6 5' splice site, and showed that mutations to this site resulted in an increase in exon 6 inclusion. Additionally, we determined that the 5' splice site of exon 6 is weak. We propose that this site may be responsible for exon 6 splicing regulation.</p><p>Using tobramycin RNA affinity chromatography followed by mass spectrometry, we identified trans-acting protein factors that bind exon 6 and regulate its splicing. These experiments identified cleavage and polyadenylation specificity factor 1 (CPSF1) among protein binding candidates. siRNA-mediated knockdown of CPSF1 resulted in an increase in exon 6 inclusion, consistent with the results of mutations to the CPSF1 binding site. Correspondingly, CPSF1 depletion had no effect on a minigene with a mutation in the intronic polyadenylation site. Finally, 3'RACE and RT-PCR experiments on RNA from Jurkat cells suggested that the intronic AAUAAA site is utilized at low frequency by the polyadenylation machinery to produce a novel polyadenylated mRNA isoform. Together, our results suggest that competing pre-mRNA splicing and polyadenylation may regulate exon 6 inclusion and resultant levels of functional IL7R produced. Since the intronic polyadenylated isoform of IL7R is predicted to be translated into a membrane-bound protein product with a shortened, signal transduction-incompetent cytoplasmic tail, this may be relevant for both T cell biology and development of multiple sclerosis.</p> / Dissertation

This dissertation focuses on the development of biological platforms on which the function and characterization of transmembrane proteins can be performed simultaneously utilizing a biomembrane mimic consisting of a solid supported phospholipid bilayer (SLB). The study centered on the platform development, biophysical measurements of transmembrane proteins and membrane species chromatography. Membrane proteins play an essential role in various cellular and physiological processes. Their normal functions are essential to our health, and many impaired proteins have been related to serious diseases. Gaining a better understanding of membrane proteins is an essential step towards the development of more specific and competent drugs. This research study is divided into two main parts. The first part centered on the creation of a new platform for allowing transmembrane proteins to freely move inside supported lipid bilayers with the same mobility that can be found in vesicle systems. SLBs have been extensively used as model systems to study cell membrane processes because they maintain the same two-dimensional fluidity of lipids within the membrane found in live cells. However, one of the most significant limitations of this platform is its inability to incorporate mobile transmembrane species. Our strategy involves supporting the lipid bilayer on a double cushion, where we not only create a large space to accommodate the transmembrane portion of the protein, but also passivate the underlying substrate to reduce non-physiological protein-substrate interactions. High diffusion constants and high mobile fractions were obtained for a transmembrane protein reconstituted within this double cushion system. The second area of this study focuses on the creation of a new method to rapidly separate membrane components using electrophoresis in SLBs. This work showed that even subtly different chemical isomers can be well-separated by a simple electrophoretic technique when cholesterol is present in the separation matrix. As a first step towards the purification of proteins, this work showed that streptavidin proteins doubly bound to a bilayer by a biotinylated lipid can be separated from streptavidin proteins which are singly bounded.

Thesis (MS)--University of Montana, 2009. / Contents viewed on November 29, 2009. Title from author supplied metadata. Includes bibliographical references.

Thesis (Ph. D.)--Syracuse University, 2009. / "Publication number:AAT 3385829."

Thesis (Ph. D.)--University OF MICHIGAN.

<p> An emerging picture in enzymology is that catalytic active site residues can perform multiple rate-enhancing features simultaneously. In triosephosphate isomerase (TIM) it is known that a glutamate acts as a general base; however, this residue may also contribute to catalysis via ground state destabilization. To systematically evaluate the contribution of the general base to both catalysis and ground state destabilization, we mutated the glutamate to uncharged polar and non-polar residues. Ablation of the general base led to a substantial 10⁴-fold decrease in activity with little catalytic variation between mutants. Binding affinity experiments revealed an increase of 10³ -fold in analog affinity for mutants lacking the charged general base supporting the presence of ground state destabilization in TIM. </p>

Part 1 of this thesis seeks to identify shared structural features of Lin28-regulated miRNAs and mRNAs. Lin28 is an evolutionarily conserved, RNA binding protein, highly expressed in stem cells and poorly differentiated cancers, that inhibits differentiation and helps maintain stem cell properties. Lin28 binds to both the loops of let-7 precursors to block let-7 biogenesis and to Lin28 responsive elements (LREs) in mRNAs either to enhance or inhibit translation. Lin28 RNA binding properties are not well defined. We used NMR spectroscopy, fluorescence assays and bioinformatics to identify common features of Lin28 targets. We show that Lin28 binds G-rich sequences that have properties of G-quartets (G4s). Based on mutational analysis, we show that G4s are important for Lin28 binding. Upon binding, Lin28 may unwind the G4 structure. Our findings suggest that Lin28 recognizes G-quartets in the RNAs it regulates and might function to unwind them. In part 2 of this thesis we use an unbiased NMR metabolite screening method to identify glucose metabolites differentially produced in BPLER and HMLER isogenic triple negative breast cancer cell lines that have dramatic differences in tumor initiating capacity. N-acetylneuraminic acid (Neu5Ac), a sugar added to the end of glycosylation chains, is much more abundant in BPLER than HMLER cells. Manipulating Neu5Ac expression using neuraminidase or siRNA knockdown of the Neu5Ac biosynthetic enzymes N-acetylneuraminic acid synthase (NANS) or cytidine monophosphate N-acetylneuraminic acid synthase (CMAS), reduces in vitro invasivity of BPLER cells. CMAS protein is also increased in BPLER relative to HMLER. Overexpressing CMAS in HMLER cells increases their invasiveness. Moreover, stable knockdown of CMAS blocks BPLER tumor growth in xenografted mice. Thus increased Neu5Ac synthesis is linked to tumor initiation and invasivity in a human triple negative breast cancer cell line.