1,720,999 research outputs found
Structural studies of CRISPR-associated proteins
No full textClustered 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
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Structural and functional studies of porins from pathogenic bacteria
Full text linkMulti-drug resistant bacteria have become a real threat to public health worldwide. Gram-negative bacteria, in particular, have shown high level of antibiotic resistance due to the presence of an additional membrane, known as outer-membrane (OM), that acts as an extra barrier. Most antibiotics enter the cells via a particular class of outer-membrane proteins (OMPs) known as porins. Porins are β-barrel channels that allow the passive diffusion of hydrophobic compounds. The porins are known to select against molecules on the basis of size and charge. When exposed to antibiotics, bacteria can modify the OM permeability by altering their porins profile. Mutations affecting the size and conductivity of the pore channel, and modification of the level of porins expression are just a few examples of how the bacteria can decrease the influx of antibiotics.
In order to better understand their interaction with antibiotics, this thesis presents structural and functional studies on porins from pathogenic bacteria. The structure of the natively expressed major outer-membrane protein (MOMP) from Campylobacter jejuni was determines, revelling the presence of a calcium-binding site inside the channel. Electro-physiology and in silico modelling analysis have shown to be important for the stability and the function of the protein. Omp50 from C. jejuni was expressed in E. Coli and its tyrosine kinase activity was analysed in vitro. Finally the structures of the two major porins from Enterobacter aerogens were determined and compared to their orthologs within the Enterobacteriaceae family. Further, a liposome-swelling assay (LSA) was used to deter-mine the rate of permeation of clinically relevant antibiotics through a series of porins. Combining these data allow a more detailed molecular understanding of translocation
Structural and functional studies of bacterial outer membrane proteins
No full textThis thesis studies two particular bacterial outer membrane proteins called OmpC and Wzi, focusing on their expression, purification, crystallization and X-ray structure determination.
A series of four naturally occurring OmpC mutants were isolated from a single patient with an E. coli infection of liver cysts. The isolated E. coli strains progressively exhibited increasing breadth of antibiotic resistance in which OmpC was predicted to take a partial role. We carried out an assay in which a strain of E. coli lacking OmpC was used to express the first (antibiotic sensitive) and the last (antibiotic resistant) of the clinical OmpC mutants and drug permeation assessed. Single channel conductance measurements were carried out and the X-ray structures for all the isolates were determined. Protein stability was assessed. With these data we propose that changes in the transverse electric field, not the pore size, underlie the clinically observed resistance to the antibiotics. This is the first demonstration of this strategy for antibiotic resistance.
Wzi is a novel outer membrane protein involved in the biosynthesis and translocation mechanism of the K30 antigen from E. coli. The mechanism is a complicated process that requires several proteins including outer and inner membrane proteins. The protein Wzi was expressed, purified and crystallized. Initial crystals were tested and diffracted to 15Å. After optimization, a crystal diffracting to 2.4Å has been obtained
Protein-carbohydrate recognition
Full text linkProtein-carbohydrate recognition is an important target for inhibitor development. Improved inhibitor design requires a fundamental molecular basis of these interactions. This thesis describes the preliminary structural studies on three carbohydrate processing enzymes, UDP-galactopyranose mutase, alpha-D-glucose-1-phosphate thymidylyltransferase and TDP-glucose 4,6-dehydratase. These enzymes are found in important human pathogens such as Mycobacterium tuberculosis and Salmonella typhimurium. The major focus of the thesis has been on UDP-galactopyranose mutase, the enzyme responsible for catalysing synthesis of the thermodynamically unfavourable 5 membered ring form of galactose, UDP-galactofuranose from the thermodynamically favoured 6 membered ring form, UDP-galactopyranose. UDP-galactofuranose plays a key role in mycobacterial cell walls. This thesis also describes work with concanavalin A. This legume lectin is an invaluable model for the study of protein-carbohydrate interactions. Two concanavalin A complexes are discussed. Both structures clear up misunderstandings in the literature and provide an insight into designing enzyme inhibitors
Structural and functional studies of bacterial outer membrane lipopolysaccharide insertion and Schmallenberg virus replication
No full textLipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of
Gram-negative bacteria and plays a fundamental role in protecting the bacteria from
harsh environments and toxic compounds. The LPS transport system is responsible
for transporting LPS from the periplasmic side of the inner membrane (IM) to the
OM, in a process involving seven LptA-LptG proteins. The current model for
lipopolysaccharide transport (Lpt) suggests that LPS is initially extracted by a four-protein complex, LptBCFG, from the inner membrane to the periplasm, where LptA
mediates further transport to the OM. Another two protein complex, LptD/E,
catalyses the assembly of LPS at the OM cell surface. However, the details of this
transport mechanism have remained unknown, mainly due to a lack of structural
information.
In chapter 1 and 2 of this thesis, I report materials and methods for all LptD/E, and
Schmallenberg virus (SBV) nucleoprotein (NP) experiments and the theories and
software that were used in determining structures of LptD/E, SBV NP and the SBV
NP/RNA complex.
In chapter 3 of this thesis, I report the first crystal structure of the outer membrane
protein LptD/E complex. LptD forms a 26-strand ß-barrel in a closed form and LptE
is a roll-like structure located inside LptD to form “barrel and plug” architecture.
Through structural analysis, function assay and molecular dynamics simulation, we
proposed a mechanism in which the hydrophilic head of LPS molecule, including the
oligosaccharide core and the O antigen, directly penetrates through the hydrophilic ß-
barrel whilst the hydrophobic lipid A tail is inserted into an intramembrane hole, with
a lateral opening between strand ß1 and ß26 of the LptD. LptE may assist this
process.
In chapter 4, I report the crystal structure of the SBV NP in two conformations:
tetrameric when the protein was purified under native conditions, and trimeric when
denatured and refolded during purification. The SBV NP has a novel fold and we
have also identified that the N-terminal arm is crucial for RNA binding, and the N- and the C-terminal arm is essential for RNA multimerisation with adjacent protomers
and for viral RNA encapsidation.
Chapter 5 describes the crystal structure of SBV NP in complex with a 42 nucleotide
long RNA (polyU). This ribonucleoprotein (RNP) complex was crystallized as a ring-like tetramer with each protomer bound to 11 ribonucleotides. Eight of these
nucleotides are bound in a positively charged cleft between N- and C- terminal
domains and three are bound in the N-terminal arm. I also compared the structure to
that of other NPs from negative-sense RNA viruses, and found that SBV NP
sequesters RNA using a different mechanism. Furthermore, the structure suggests that
when RNA binds the protein, there are conformational changes in the RNA-binding
cleft, and in the N- and C-terminal arms. Thus our results reveal a novel mechanism
of RNA encapsidation by orthobunyaviruses NP
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Structural analysis of the potential therapeutic targets from specific genes in Methicillin-resistant Staphylococcus aureus (MRSA)
No full textThe thesis describes over-expression, purification and crystallization of three proteins from Staphylococcus aureus (S. aureus). S. aureus is an important human pathogen and methicillin-resistant S. aureus (MRSA) is a serious problem in hospitals nowadays. The crystal structure of 3-Methyladenine DNA glycosylase I (TAG) was determined by single-wavelength anomalous diffraction (SAD) method. TAG is responsible for DNA repair and is an essential gene for both MRSA and methicilin-susceptible S. aureus (MSSA). The structure was also determined in complex with 3-methyladenine (3-MeA) and was solved using molecular replacement (MR) method. An assay was carried out and the molecular basis of discrimination between 3-MeA and adenosine was determined.
The native crystal structure of fructose 1-phosphate kinase (PFK) from S. aureus was determined to 2.30 Å and solved using molecular replacement method. PFK is an essential enzyme involved in the central metabolism of MRSA. Despite extensive efforts no co-complex was determined, although crystals were obtained they diffracted poorly. An assay which can be used to test for inhibitors has been developed.
Mevalonate Kinase (MK) is another essential enzyme in MRSA and is a key drug target in the mevalonate pathway. Native data diffracting to 2.2 Å was collected. The structure was solved using multiple isomorphorus replacement (MIR) method. A citrate molecule was bound at the MK active site, arising from the crystallization condition. The citrate molecule indicates how substrate might bind. The protein was kinetically characterized. A thermodynamic analysis using fluorescence-based method was carried out on each protein to investigate binding interactions of potential fragments and thus a drug design starting point
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