2024-03-29T13:25:42Zhttps://research-repository.st-andrews.ac.uk/oai/requestoai:research-repository.st-andrews.ac.uk:10023/5422019-07-01T10:16:27Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Functional analysis of the orthobunyavirus nucleocapsid (N) protein
Eifan, Saleh A.
Elliott, Richard Michael
Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae. It has a
tripartite genome consisting of negative sense RNA segments called large (L),
medium (M) and small (S). The S segment encodes the nucleocapsid protein (N) of
233 amino acids. The N protein encapsidates all three segments to form
transcriptionally active ribonucleoproteins (RNPs). The aim of this project was to
determine the domain map of BUNV N protein.
To investigate residues in BUNV N crucial for its functionality, random and site-
specific mutagenesis were performed on a cDNA clone encoding the BUNV N
protein. In total, 102 single amino acid substitutions were generated in the BUNV N
protein sequence.
All mutant N proteins were used in a BUNV minigenome system to compare their
activity to wt BUNV N. The mutant proteins displayed a wide-range of activity, from
parental-like to essentially inactive. The most disruptive mutations were R94A,
I118N, W134A, Y141C, L177A, K179I and W193A.
Sixty-four clones carrying single substitutions in the BUNV N protein were used in the
BUNV rescue system in an attempt to recover viable mutant viruses. Fifty
recombinant mutant viruses were rescued and 14 N genes were nonrescuable.
The 50 mutant viruses were characterized by: titration, protein labelling, western
blotting, temperature sensitivity and host-restriction. Mutant viruses displayed a wide range of titers between 10³
-10⁸ pfu/ml, and three different plaque sizes large,
medium and small. Protein labelling and western blotting showed that mutations in
the N gene did not affect expression of the other viral genes as much as affecting N
protein expression. It was demonstrated that single amino acid substitutions could
alter N protein electrophoretic mobility in SDS- PAGE (e.g. P19Q and L53F).
Temperature sensitivity tests showed that recombinant viruses N74S, S96S, K228T
and G230R were ts, growing at 33˚C but not at 37˚C or 38˚C, while the parental virus grew at all temperatures. Using the northern blotting technique, mutant viruses N74S
and S96G were shown to have a ts defect in genome-synthesis (late replication
step), while mutant viruses K228T and G230R had a ts defect in antigenome-
synthesis (early replication step).
Host-restriction experiments were performed using 5 different cell lines (Vero-E6,
BHK-21, 2FTGH-V, A549-V and 293-V). Overall, the parental virus grew similarly in
all cell lines. Likewise, the majority of mutant viruses follow this pattern except mutant
virus Y23A. It showed a 100-fold reduction in titer in 2FTGH-V cells. Comparing the
ratios of intracellular and extracellular particles revealed that only 15% of the total
virus particles of mutant Y23A was released as extracellular particles compared to
30% of the parental virus.
Fourteen N genes were nonrescuable. They were characterized by (i) their activity in
the BUNV minigenome system, (ii) their activity in BUNV packaging assay, (iii) their
ability to form multimers, (iv) their ability to interact with L protein, and (v) their impact
on RNA synthesis.
In summary, BUNV N protein was shown to be multi-functional and involved in the
regulation of virus transcription and replication, RNA synthesis and assembly, via
interactions with the viral L polymerase, RNA backbone, itself or the viral
glycoproteins.
2008-10-27
2008-10-27
2008
Thesis
http://hdl.handle.net/10023/542
en
http://creativecommons.org/licenses/by-nc-nd/3.0/
Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported
xviii, 225 p.
University of St Andrews
The University of St Andrews
oai:research-repository.st-andrews.ac.uk:10023/39652019-03-29T11:19:48Zcom_10023_175com_10023_39com_10023_51com_10023_18col_10023_177col_10023_53
Structural studies of CRISPR-associated proteins
Reeks, Judith
Naismith, Jim
X-ray crystallography
CRISPR/Cas
S. solfataricus
FaNaC
ASIC
Clustered regularly interspaced short palindromic repeats (CRISPRs) act to prevent viral infection and horizontal gene transfer in prokaryotes. The genomic CRISPR array contains short sequences (“spacers”) that are derived from foreign genetic elements. The CRISPR array is transcribed and processed into CRISPR RNAs (crRNAs) used in the sequence-specific degradation of foreign nucleic acids. This process is called interference and is mediated by CRISPR-associated (Cas) proteins.
This thesis has focused on the structural and functional characterisation of four Cas proteins from the CRISPR/Cas system of Sulfolobus solfataricus. The crystal structure of Cmr7 (Sso1725), a Sulfolobales-specific subunit of the ssRNA-degrading CMR complex, allowed for the identification of a putative protein-binding site, though no specific function could be ascribed to the protein. Cas6 (Sso1437) is the enzyme responsible for crRNA maturation and the characterisation of this protein allowed for the molecular rationalisation of its atypical RNA cleavage mechanism. Csa5 and Cas8a2 are subunits of the aCascade complex that targets dsDNA. Csa5 (Sso1398) was shown to have a putative role in R-loop stabilisation during interference while the role of Cas8a2 (Sso1401) was not determined. The structures of these two proteins were used to define relationships between the subunits of interference complexes from various CRISPR/Cas systems.
A second aspect of this work has been the expression and purification of eukaryotic ion channels for structural studies. The acid sensing ion channel (ASIC) and FMRFamide-gated sodium channel (FaNaC) are gated ion channels with unknown mechanisms of channel activation. These ion channels must be expressed in eukaryotic systems and so human embryonic kidney (HEK) cells and baculovirus-insect cell expression systems were developed to express ASIC and FaNaC constructs. The expression and purification protocols have been optimised to allow for the preparation of soluble protein that will in future be used for crystallography and electron paramagnetic resonance (EPR) studies.
2013-08-16
2013-08-16
2013-11-29
Thesis
http://hdl.handle.net/10023/3965
http://hdl.handle.net/10023/3954
http://hdl.handle.net/10023/3863
http://hdl.handle.net/10023/3550
http://hdl.handle.net/10023/5991
en
Reeks , J , Sokolowski , R D , Graham , S , Liu , H , Naismith , J H & White , M F 2013 , ' Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing ' Biochemical Journal , vol 452 , no. 2 , pp. 223-230
Reeks , J A , Naismith , J & White , M F 2013 , ' CRISPR interference : a structural perspective ' Biochemical Journal , vol 453 , no. 2 , pp. 155-166
Reeks , J A , Graham , S , Anderson , L , Liu , H , White , M F & Naismith , J 2013 , ' Structure of the archaeal Cascade subunit Csa5 : Relating the small subunits of CRISPR effector complexes ' RNA Biology , vol 10 , no. 5 , pp. 762-769
Zhang , J , Rouillon , C , Kerou , M , Reeks , J , Brugger , K , Graham , S , Reimann , J , Cannone , G , Liu , H , Albers , S-V , Naismith , J H , Spagnolo , L & White , M F 2012 , ' Structure and mechanism of the CMR complex for CRISPR-Mediated antiviral immunity ' Molecular Cell , vol 45 , no. 3 , pp. 303-313
330
University of St Andrews
The University of St Andrews
oai:research-repository.st-andrews.ac.uk:10023/103592017-02-24T10:28:06Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Structure and function of heparan sulfate degrading sulfatases
Griffin, Laura Susan
Gloster, Tracey
Biotechnology and Biological Sciences Research Council (BBSRC)
Heparan sulfate
Sulfatases
2017-02-24
2017-02-24
2017-06-21
Thesis
http://hdl.handle.net/10023/10359
en
http://creativecommons.org/licenses/by-nc-nd/4.0/
2019-02-03
Thesis restricted in accordance with University regulations. Print and electronic copy restricted until 3rd February 2019
Attribution-NonCommercial-NoDerivatives 4.0 International
278 p.
University of St Andrews
The University of St Andrews
oai:research-repository.st-andrews.ac.uk:10023/63192019-03-29T10:30:05Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Molecular studies of the Tacaribe virus nucleoprotein (NP): identification and characterisation of virus-host interactions as novel anti-arenavirus drug targets
Meyer, Bjoern
Jackson, David
Medical Research Council (MRC)
Virology
Virus
Arenavirus
Tacaribe virus
Innate immunity
High throughput screen
Reverse genetics
Interferon
Protein-protein interaction
Arenaviruses cause an estimated 300,000 – 500,000 infections annually. Currently there is no arenavirus-specific antiviral drug available to treat these infections. This study sought to use the non-pathogenic New World arenavirus Tacaribe virus (TCRV) as a model for the pathogenic Junin virus (JUNV) and Machupo virus (MACV) that cause haemorrhagic fevers in South America. TCRV was used to explore three different approaches in the search for an antiviral drug against arenavirus infection targeted specifically against the viral nucleoprotein (NP). Of the four expressed arenaviral proteins, NP is the most abundant and is thought to be of multifunctional nature involved in viral replication, suppression of the innate immune system and viral egress. The approaches to find targets for broad-spectrum anti-arenaviral drugs were high throughput screens (HTS) with purified NP using thermal shift assays, exploring the virus interactions with the innate immune system and identifying virus-host protein-protein interactions. HTS resulted in the identification of two small-molecule compounds, [5-(2-Furyl)thien-2-yl]methanol and cyclosporine A (CsA), showing broad-spectrum activity against arenaviruses. Interferon-stimulated genes (ISGs), such as IFIT3, were identified to reduce viral titres and potential 202 protein-interactions between NP and host cell proteins were identified, of which the interaction with apoptosis-inducing factor 1 (AIF1) was described further. To characterise the importance of these interactions as potential drug targets further, a TCRV reverse genetics system was constructed.
2015-03-25
2015-03-25
2014
Thesis
http://hdl.handle.net/10023/6319
en
vi, 317 p.
University of St Andrews
The University of St Andrews
oai:research-repository.st-andrews.ac.uk:10023/8812019-07-01T10:12:46Zcom_10023_175com_10023_39col_10023_177
Effects of cationic antimicrobial peptides on Candida and Saccharomyces species
Harris, Mark R.
Coote, Peter John
Biotechnology and Biological Sciences Research Council (BBSRC)
Antimicrobial peptide
Echinocandin
Candida
Saccharomyces
Electronic version does not contain associated previously published material
Antimicrobial peptides (AMPs) are found throughout the animal kingdom and act as a natural defence against a broad spectrum of pathogens. These peptides are toxic to invading organisms without acting on host cells, so are of interest for their potential to act as potent new drugs against pathogenic organisms. AMPs traverse the cell wall and predominantly target the plasma membrane, resulting in destabilisation, leakage of intracellular components and cell death. In this thesis the mode of action of several AMPs was investigated. The role of the cell wall was studied and found to mediate peptide binding, the inhibition of certain cell wall components also increased peptide action, subsequent internalisation events were observed with varying localisation patterns and the effect of several genes that alter cell susceptibility to AMP were examined.
Several Candida albicans mutants, each deficient in cell wall protein mannosylation, were tested in relation to their susceptibility to AMPs. It was discovered that cells lacking or deficient in the phosphomannan fraction, with a concomitant reduction in surface negative charge, correlated with reduced susceptibility to AMP action. To ascertain whether peptide binds to negatively charged phosphate, the effect of exogenous glucosamine 6-phosphate (but not glucosamine hydrochloride) was studied demonstrating that peptide efficacy was reduced due to the presence of exogenous phosphate. More specifically, sequestration of the truncated cationic AMP dermaseptin S3 (DsS3(1-16)) was reduced in these phosphomannan deficient mutants. Microscopy analysis of fluorescein tagged DsS3(1-16) also revealed the differential localisation patterns of this AMP: transiently binding to the plasma membrane, localisation to the vacuole or diffuse distribution throughout the cytoplasm. It is proposed that for these cationic AMPs to exert their full antifungal action they must first bind to the negatively charged phosphate.
The echinocandins are a relatively new class of antifungal that function by inhibiting 1,3-β glucan synthase resulting in reduced 1,3-β glucan in the cell wall. As AMPs have to traverse the cell wall it was postulated that cells lacking this fraction would display increased AMP binding to the membrane. Clinical isolate strains of Candida and Cryptococcus spp. were acquired to test their susceptibility to AMP and echinocandin combinations. Comparing the fractional inhibitory concentration index (FICI) (supported by viable cell counts and on a solid surface using disc diffusion assays) synergy was observed between caspofungin, anidulafungin and several AMPs in vitro. In vitro toxicity assays revealed no increase in haemolytic or cytotoxic action on combination. These synergistic combinations could provide a novel treatment against fungal pathogens.
The final area of study was based upon work that identified genes whose expression altered cell susceptibility to AMPs. Three genes were selected for investigation that upon deletion increased the action of DsS3(1-16) or magainin 2 on S. cerevisiae. Results from growth analysis, peptide sequestration and cell viability counts confirmed that deletion of HAL5, LDB7 or IMP2’ did increase susceptibility. Additionally, deletion of HAL5 increased the probability of cell depolarisation upon peptide exposure. Expression of GFP-tagged Imp2’ also increased when cells were exposed to DsS3(1-16). It was concluded that deletion of HAL5 increases depolarisation due to insufficient potassium efflux, leading to ion leakage and cell death facilitated by AMP action. Double strand break repair and DNA protection are probably compromised upon deletion of LDB7 and IMP2’, increasing the inhibitory action of DsS3(1-16) that has previously been shown to bind to DNA.
2010-05-26
2010-05-26
2010-06-23
Thesis
http://hdl.handle.net/10023/881
en
http://creativecommons.org/licenses/by/3.0/
Creative Commons Attribution 3.0 Unported
266
University of St Andrews
The University of St Andrews
oai:research-repository.st-andrews.ac.uk:10023/169832019-07-01T10:15:15Zcom_10023_175com_10023_39com_10023_51com_10023_18col_10023_177col_10023_53
Lipidomic investigations into the phospholipid content and metabolism of various kinetoplastids
Roberts, Matthew D.
Smith, Terry K.
Wellcome Trust
Lipidomic
Mass spectrometry
Typanosoma brucei
Leishmania
Crithidia fasiculata
Phospholipid
Fatty acid
Phosphonolipid
Cyclopropyl fatty acid
Kinetoplastid
Parasite
Model organism
Metabolism
Lipid
Typanosoma cruzi
This work expands the knowledge on phospholipid metabolism in the kinetoplastid
parasites: T. brucei, T. cruzi, Leishmania spp. that cause neglected tropical diseases
and the related non-human pathogenic Crithidia fasiculata.
As a close relative of parasitic kinetoplasts, specifically Leishmania, it is hypothesised
that Crithidia fasiculata possesses a similar lipid biosynthetic capability and therefore
represent an attractive model organism. Database mining the Crithidia genome
revealed the ability to biosynthesise all of the main phospholipid species. Utilising
various lipidomic techniques, a high level of an ω-6 18:3 fatty acid was observed,
alongside an uncommon Δ19:0 fatty acid that was later identified to be exclusive
attributed to PE species. Sphingolipid metabolism was shown to resemble that of
Leishmania and T. cruzi, given the exclusive production of inositol-phosphoceramide
species and no sphingomyelin species being observed. Using labelled precursors,
Crithidia were seen to uptake and incorporate extracellular inositol into both
phosphatidylinositol and inositol-phosphoceramide species. Crithidia were also
shown to utilise both the Kennedy pathway and methylation of
phosphatidylethanolamine to form phosphatidylcholine.
The phospholipidome of T. cruzi revealed several phosphatidylserine species for the
first time, suggesting a functional phosphatidylserine synthase. Current knowledge
of T.cruzi sphingolipid biosynthesis was also confirmed as only inositol
xxxi
phosphoceramide species were observed. The identification and subsequent
characterisation of novel phosphonolipid species are reported for the first time.
Utilising lipidomic methodologies and labelled precursors, the relative contribution
of the intracellular inositol pools within bloodstream and procyclic T. brucei towards
PI biosynthesis was examined. This highlighted that the synthesis/turnover rates for
specific phosphatidylinositol and inositol-phosphoceramide species are unequal.
Efforts to optimise media conditions highlighted that under reduced levels of
serum/glucose/inositol, bloodstream T. brucei unexpectedly adjusts its inositol
metabolism. The procyclic parasite exemplifies this fact, as under inositol/glucose
deficient media conditions they appear to have adapted to utilising glucogenesis and
inositol de-novo synthesis.
This work highlights that these parasites are rapidly dividing, their unique features of
lipid metabolism may be exploitable for drug discovery purposes.
2019-02-01
2019-02-01
2017-06-21
Thesis
http://hdl.handle.net/10023/16983
en
http://creativecommons.org/licenses/by-nc-nd/4.0/
2020-04-05
Thesis restricted in accordance with University regulations. Electronic copy restricted until 5th April 2020
Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
xxxi, 331 p.
University of St Andrews
The University of St Andrews
oai:research-repository.st-andrews.ac.uk:10023/122732017-12-06T14:36:30Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Title redacted
Jameison, Stephen A.
Taylor, Garry L.
Biotechnology and Biological Sciences Research Council (BBSRC)
University of St Andrews. School of Biology
2017-12-06
2017-12-06
2017-12-07
Thesis
http://hdl.handle.net/10023/12273
en
2019-11-22
Thesis restricted in accordance with University regulations. Print and electronic copy restricted until 22nd November 2019
xxxix, 552 p.
University of St Andrews
The University of St Andrews
Biomedical Sciences Research Complex (BSRC)
oai:research-repository.st-andrews.ac.uk:10023/4682019-07-01T10:18:35Zcom_10023_175com_10023_39col_10023_177
Characterisation of Sulfolobus solfataricus Ard1, a promiscuous N-acetyltransferase
Mackay, Dale Tara
White, Malcolm F.
Taylor, Garry L.
Biotechnology and Biological Sciences Research Council (BBSRC)
Sulfolobus
Acetylation
Compaction of DNA into chromatin is an important feature of every living cell. This compaction phenomenon is brought about and maintained by a variety of DNA binding proteins, which have evolved to suit the specific needs of the different cell types spanning the three kingdoms of life; the eukaryotes, prokaryotes and archaea. Sulfolobus solfataricus, a member of the crenarchaeal subdivision of the archaea, has two prominent DNA binding proteins known as Alba (1&2) and Sso7d. Alba1 is acetylated in vivo at two positions and this modification lowers its’ affinity for binding DNA. Acetylation levels impact many cellular processes and in higher organisms play a critical role in the development of many cancers and other diseases.
This thesis documents the finding and characterisation of the N-terminal acetyltransferase (ssArd1) of SsoAlba1, based on its’ sequence homology to the catalytic subunits Ard1, Nat3 and Mak3 belonging to the larger eukaryal Nat complexes NatA, NatB and NatC, respectively. Mutagenesis studies revealed that ssArd1 preferentially acetylates N-termini bearing a serine or alanine residue at position 1 (after methionine cleavage). It is also capable of acetylating other proteins with very different physical structures. These findings allow classification of ssArd1 as a promiscuous acetyltransferase belonging to the Gcn5-N-acetyltransferase (GNAT) superfamily. The active site of the enzyme was examined through mutagenesis studies, revealing that the mechanism of acetylation is likely to proceed through a direct acetyl transfer involving a tetrahedral intermediate. Structural studies provided some insight into the molecular structure of ssArd1.
2008-04-23
2008-04-23
2008-06-27
Thesis
http://hdl.handle.net/10023/468
en
http://creativecommons.org/licenses/by/3.0/
Creative Commons Attribution 3.0 Unported
209
University of St Andrews
The University of St Andrews
oai:research-repository.st-andrews.ac.uk:10023/44222019-07-01T10:19:02Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Structural and functional characterisation of Mcb1 and the MCMᴹᶜᵇ¹ complex in Schizosaccharomyces pombe
Schnick, Jasmin
MacNeill, Stuart
DNA replication
Minichromosome maintenance binding protein
Schizosaccharomyces pombe
Mcm2-7 complex
Protein interaction
DNA damage checkpoint
Temperature-sensitive mutants
The MCM helicase plays an important role in eukaryotic DNA replication, unwinding double stranded DNA ahead of the replication fork. MCM is a hetero-hexamer consisting of the six related proteins, Mcm2-Mcm7. The distantly related MCM-binding protein (MCM-BP) was first identified in a screen for proteins interacting with MCM2-7 in human cells and was found to specifically interact with Mcm3-7 but not Mcm2. It is conserved in most eukaryotes and seems to play an important role in DNA replication but its exact function is not clear yet.
This study contributes to the understanding of the fission yeast homologue of MCM-BP, named Mcb1, but also of MCM-BP in general. Results presented in this thesis document the initial biochemical characterisation of the complex Mcb1 forms with Mcm proteins, the MCMᴹᶜᵇ¹ complex. Interactions of Mcb1 with Mcm proteins, potential interaction sites between the proteins and the size of the complex were analysed using a variety of methods, including tandem affinity purification, co-immunoprecipitation, sucrose gradients and in vitro pull-down assays. Sequence analysis and structure prediction were utilised to gain some insight into Mcb1 and MCM-BP ancestry and structure. Results presented here indicate that fission yeast Mcb1 shares homology with Mcm proteins and forms a complex with Mcm3-Mcm7 but not Mcm2 and thus replaces the latter in an alternative high molecular weight complex that is likely to have an MCM-like appearance.
Deletion of mcb1⁺ showed that Mcb1 is essential in fission yeast. To examine the cellular function of the protein, temperature-sensitive mutants were generated. Inactivation of Mcb1 leads to an increase in DNA damage and cell cycle arrest in G2-phase depending on the activation of the Chk1 dependent DNA damage checkpoint. Similar observations were made when Mcb1 was overexpressed, indicating that certain levels of the protein are important for accurate DNA replication. Construction of truncated versions of Mcb1 suggested that almost the full-length protein is needed for proper function.
2014-01-28
2014-01-28
2014-06
Thesis
http://hdl.handle.net/10023/4422
en
http://creativecommons.org/licenses/by-sa/4.0/
Creative Commons Attribution-ShareAlike 4.0 International
xi, 267 p.
University of St Andrews
The University of St Andrews
oai:research-repository.st-andrews.ac.uk:10023/45062019-03-29T10:37:03Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Structural studies on human dihydroxyacetone kinase (DAK) and the carbohydrate-binding domain of Streptococcus pneumoniae NanA
Yang, Lei
Taylor, Garry L.
A number of dihydroxyacetone kinases (DAK) have been described that can utilize
either ATP (in animals, plants and some bacteria) or phosphoenolpyruvate (PEP) (in
most bacteria) as the energy source to convert dihydroxyacetone (Dha) to
dihydroxyacetone phosphate (DhaP), which plays critical roles in many metabolic
pathways. It has also been described that Homo sapiens DAK is able to regulate the
innate immune system via its interaction with Melanoma differentiation-associated
protein 5 (MDA5), which is recognized as an RNA sensor during virus infection.
These findings make H. sapiens DAK a very noteworthy research target due to its
multiple functions in many fields. Therefore, structural studies of DAK from H.
sapiens are presented.
The initial structure of the wild type (WT) H. sapiens DAK was determined to 2.5Å
resolution and solved by molecular replacement. The structure forms a homodimer
and four dimers were shown to be present in each asymmetric unit. However, within
each monomer, most regions of the C-terminal domain were disordered, and therefore
in order to improve structure quality, multiple site-directed mutagenesis was used. The
mutated protein was then crystallized and the structure was determined to 1.4Å. The
N-terminal Dha binding domain consists of two α/β regions and the C-terminal ATP
binding domain is comprised of eight anti-parallel α-helices, which forms a deep
pocket and is filled with a phospholipid molecule. In addition, the structures of
mutated DAK in complex with ATP analogues and Dha are also described in the
current study.
The second part of the project concerned sialidases, which are glycoside hydrolases that specifically hydrolyse terminal sialic acid from various glycans. Streptococcus
pneumoniae is one of the most common pathogenic bacteria of humans, and is
reported to encode three sialidases that act as virulence factors in bacterial
colonization and infection. One of these sialidases, NanA, was reported to be present
in all clinical strains and plays a vital role during the bacterial infection.
Consequently, the structure of N-terminal Carbohydrate-binding module (CBM)
domain of NanA has been determined to 1.8Å, and reveals a β-sandwich fold. The apo
form of NanA-CBM is present as a dimer in the asymmetric unit, whereas a monomer
was detected when it is bound to sialic acid or its derivatives. Structural comparisons
between NanA-CBM and other structures of the CBM40 family were also performed.
The substrate binding sites of NanA-CBM forms a cavity that is able to accommodate
the substrates. A potential molecular binding site located beside the sialic acid binding
site was revealed, and is occupied by the side chain of a lysine from a symmetry-
related molecule. Heteronuclear single quantum coherence (HSQC) NMR
spectroscopy and fluorescent-based thermal shift assays were also carried out to
further characterise the protein.
The current results reveal the structure of both DAK from H. sapiens and NanA-CBM
from S. pneumoniae, which may contribute to a better understanding towards cell
metabolism and bacterial colonization.
2014-03-11
2014-03-11
2013
Thesis
http://hdl.handle.net/10023/4506
en
xvii, 171 p.
University of St Andrews
The University of St Andrews
oai:research-repository.st-andrews.ac.uk:10023/102542017-02-09T10:24:15Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Structural and mechanistic studies of specific carbohydrate processing enzymes
Pengelly, Robert Joseph
Gloster, Tracey
Wellcome Trust
2017-02-09
2017-02-09
2017-06-21
Thesis
http://hdl.handle.net/10023/10254
en
http://creativecommons.org/licenses/by-nc-nd/4.0/
2019-02-03
Thesis restricted in accordance with University regulations. Print and electronic copy restricted until 3rd February 2019
Attribution-NonCommercial-NoDerivatives 4.0 International
xxiii, 297 p.
University of St Andrews
The University of St Andrews
oai:research-repository.st-andrews.ac.uk:10023/32302019-03-29T10:37:43Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Attenuation of bunyavirus replication by modification of genomic untranslated regions
Mazel-Sanchez, Beryl
Elliott, Richard Michael
Bunyamwera orthobunyavirus (BUNV) is the prototype for the family Bunyaviridae.
BUNV has a tripartite RNA genome of negative polarity composed of the large(L),medium (M)and small(S)segments. Each segment contains an open reading frame (ORF) flanked by untranslated regions (UTRs). The eleven terminal nucleotides are conserved between the three segments while the internal regions are unique. The UTRs play an important role in the virus life cycle by promoting transcription, replication and encapsidation of the viral genome.The work presented in this thesis explores UTRs plasticity and examines ways to engineere attenuated viruses by modifying only their UTRs. Using reverse genetics, mainly two ways of attenuation were explored: rescue of viruses either carrying deletions within their 3’ and/or 5’ UTRs in all three segments, or of viruses carrying one segment bearing heterologous UTRs. Both approaches resulted in virus attenuation in tissue culture, with viruses producing smaller plaques or even no plaques, and growing to lower titres than wild-type BUNV. Through serial passage, viruses were shown to
regain some level of fitness while the mutations introduced in the UTRs proved to be stable. Thus, to investigate the mechanism behind fitness recovery, the nucleotide sequence of the entire genome of viruses with deletions in their UTRs was determined. Amino acid changes were observed in the viral polymerase (L protein) of most mutant viruses and the vast majority of the amino acid changes occured in the C-terminal region. The function of this domain is unclear to date, however data obtained using a mini-replicon assay suggest that the
C-terminal domain of the L protein might be involved in UTR recognition. Full genome
sequencing also allowed the identification of an amino acid mutation within the polymerase that resulted in a temperature sensitive phenotype when introduced in an otherwise wild-type BUNV. Thus, it was shown that mutations introduced within the UTR regions of the genome were stable through serial passage and resulted in attenuation. Such a strategy could be used in
combination with mutations of the ORF to design live-attenuated vaccines against serious pathogens within the family Bunyaviridae.
2012-10-25
2012-10-25
2012-11-30
Thesis
http://hdl.handle.net/10023/3230
en
214
University of St Andrews
The University of St Andrews
oai:research-repository.st-andrews.ac.uk:10023/25702019-07-01T10:11:49Zcom_10023_175com_10023_39col_10023_177
Replication of Bunyamwera virus in mosquito cells
Szemiel, Agnieszka M.
Elliott, Richard Michael
University of St Andrews
The Bunyaviridae family is one of the largest among RNA viruses, comprising more
than 350 serologically distinct viruses. The family is classified into five genera,
Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus.
Orthobunyaviruses, nairoviruses and phleboviruses are maintained in nature by a
propagative cycle involving blood-feeding arthropods and susceptible vertebrate hosts.
Like most arthropod-borne viruses, bunyavirus replication causes little damage to the
vector, whereas infection of the mammalian host may lead to death. This situation is
mimicked in the laboratory: in cultured mosquito cells no cytopathology is observed
and a persistent infection is established, whereas in cultured mammalian cells
orthobunyavirus infection is lytic and leads to cell death.
Bunyaviruses encode four common structural proteins: an RNA-dependent RNA
polymerase, two glycoproteins (Gc and Gn), and a nucleoprotein N. Some viruses also
code for nonstructural proteins called NSm and NSs. The NSs protein of the prototype
bunyavirus, Bunyamwera virus, seems to be one of the factors responsible for the
different outcomes of infection in mammalian and mosquito cell lines. However, only
limited information is available on the growth of bunyaviruses in cultured mosquito cell
lines other than Aedes albopictus C6/36 cells. Here, I compared the replication of
Bunyamwera virus in two additional Aedes albopictus cell clones, C7-10 and U4.4, and
two Aedes aegypti cell clones, Ae and A20, and investigated the impact of virus
replication on cell function. In addition, whereas the vertebrate innate immune
response to arbovirus infection is well studied, relatively little is known about
mosquitoes’ reaction to these infections. I investigated the immune responses of the
different mosquito cells to Bunyamwera virus infection, in particular antimicrobial
signaling pathways (Toll and IMD) and RNA interference (RNAi). The data obtained in
U4.4 cells suggest that NSs plays an important role in the infection of mosquitoes.
Moreover infection of U4.4 cells more closely resembles infection in Ae and A20 cells
and live Aedes aegypti mosquitoes. My data showed that the investigated cell lines
have various properties, and therefore they can be used to study different aspects of
mosquito-virus interactions.
2012-04-24
2012-04-24
2011
Thesis
http://hdl.handle.net/10023/2570
en
http://creativecommons.org/licenses/by-nc-nd/3.0/
Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported
University of St Andrews
The University of St Andrews
oai:research-repository.st-andrews.ac.uk:10023/5712019-07-01T10:18:00Zcom_10023_175com_10023_39com_10023_51com_10023_18col_10023_177col_10023_53
The synthesis of 5-substituted hydantoins
Murray, Ross George
Conway, Stuart J.
sanofi-aventis
Hydantoin
Aminonitrile
Synthesis
Lewis acid catalysis
The Bucherer-Bergs reaction is a classical multi-component reaction that yields hydantoins, which can be hydrolysed to afford α-amino acids. Hydantoins have many uses in modern organic synthesis, and this moiety has been included in a number of therapeutic agents, which have a wide range of biological activities. Herein, we report a mild synthesis of 5- and 5,5-substituted hydantoins from α-aminonitriles using Hünig’s base and carbon dioxide. This reaction can be performed in excellent yields, using a variety of organic solvents and is applicable to a range of substrates. In an extension to the above methodology, a one-pot Lewis acid-catalysed synthesis of hydantoins from ketones has also been developed and optimised in organic media. This reaction can be performed in excellent yields and is suitable for the synthesis of 5- and 5,5-substituted hydantoins.
2008-12-02
2008-12-02
2008-06-25
Thesis
http://hdl.handle.net/10023/571
en
http://creativecommons.org/licenses/by-nc-nd/3.0/
Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported
192
University of St Andrews
The University of St Andrews
oai:research-repository.st-andrews.ac.uk:10023/36802019-03-29T10:41:20Zcom_10023_45com_10023_17com_10023_175com_10023_39col_10023_47col_10023_177
Distance measurements using pulsed EPR : noncovalently bound nitroxide and trityl spin labels
Reginsson, Gunnar Widtfeldt
Schiemann, Olav
Sigurdsson, Snorri T.
EPR
PELDOR
DEER
Distance measurements
Free radicals
Spin labels
Nitroxide radicals
Trityl radicals
Biomolecular structure
Nucleic acid structure
The function of biomacromolecules is controlled by their structure and conformational flexibility. Investigating the structure of biologically important macromolecules can, therefore, yield information that could explain their complex biological function. In addition to X ray crystallography and nuclear magnetic resonance (NMR) methods, pulsed electron paramagnetic resonance (EPR) methods, in particular the pulsed electron electron double resonance (PELDOR) technique has, during the last decade, become a valuable tool for structural determination of macromolecules. Long range distance constraints obtained from pulsed EPR measurements, make it possible to carry out structural refinements on structures from NMR and X ray methods. In addition, EPR yields distance distributions that give information about structural flexibility.
The use of EPR for structural studies of biomacromolecules requires in most cases site specific incorporation of paramagnetic centres known as spin labelling. To date, spin labelling nucleic acids has required complex spin labelling chemistry. The first application of a site directed and noncovalent spin labelling method for distance measurements on DNA is described. It is demonstrated that noncovalent spin labelling with a rigid spin label can afford detailed information on internal DNA dynamics using PELDOR. Furthermore, it is shown that noncovalent spin labelling can be used to study DNA protein complexes.
PELDOR can also yield information about spin label orientation. Therefore, spin labels with limited flexibility can be used to measure the relative orientation of the spin labelled sites. Although information on orientation can be obtained from 9.7 GHz PELDOR measurements in selected applications, measurements at 97 GHz or higher, increases orientation selection. It is shown that PELDOR measurements on semi rigid and rigid nitroxide biradicals using a home built high power 97 GHz EPR spectrometer (Hiper) and model based simulations yield quantitative information on spin label orientations and dynamics.
The most widely used spin labels for EPR studies on biomacromolecules are the aminoxyl (nitroxide) radicals. The major drawbacks of nitroxide spin labels include low sensitivity for distance measurements, fast spin spin relaxation in solution and limited stability in reducing environments. Carbon centered triarylmethyl (trityl) radicals have properties that could eliminate some of the limitations of nitroxide spin labels. To evaluate the use of trityl spin labels for nanometer distance measurements, models systems with trityl and nitroxide spin labels were measured using PELDOR and Double Quantum Coherence (DQC). This study shows that trityl spin labels yield reliable information on interlabel distances and dynamics, establishing the trityl radical as a viable spin label for structural studies on biomacromolecules.
2013-06-13
2013-06-13
2013-06-26
Thesis
http://hdl.handle.net/10023/3680
en
372
University of St Andrews
The University of St Andrews
University of Iceland