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A computational swiss army knife approach to unravelling the secrets of proton movement through SERCA
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Structural and biophysical characterisation of the extra-cellular domains from the mammalian peptide transporters, PepT1 and PepT2
Full text linkPepT1 and PepT2 are integral membrane proteins which couple the uptake of di- and tri-peptides to the proton electro-chemical gradient. PepT1 is predominantly expressed in the small intestine and is the main route through which dietary protein is absorbed. PepT2 shares 46% sequence identity with PepT1 and is expressed in the kidneys, lung and central nervous system. Many commonly prescribed drugs, such as penicillin are peptide mimetics, and PepT1 and PepT2 play a direct role in their transport and pharmacokinetic properties. Recent X-ray crystal structures and functional studies of the bacterial peptide transporters have provided significant insight into the likely mechanism by which such drugs are recognised by PepT1 and PepT2. The bacterial peptide transporters share approximately 30% sequence identity within their trans-membrane domains to the mammalian PepT1 and PepT2 transporters. However, a key structural difference exists; an additional 20 kDa extra-cellular loop is inserted between trans-membrane helices 9 and 10 in the animal peptide transporters, and this loop is absent in the bacterial homologues. It was not known, prior to this thesis, if this extra-cellular loop was structured and/or integral to the transport cycle, or whether it served an additional function to assist or regulate peptide transport. To investigate the role of this domain, the crystal structures of the Mus musculus PepT1 and Rattus norvegicus PepT2 'loops' were determined to 2.10 and 2.06 Å resolution respectively. The structures indicated that the loop region in both PepT1 and PepT2 forms a bi-lobal, all β-sheet, self-contained extra-cellular domain (ECD). Despite low sequence similarity, the ECDs from PepT1 and PepT2 share a common architecture; two transthyretin-like folds connected by a flexible linker. Sequence and structural analyses have indicated that the lobe interface of MmPepTECD is maintained by two highly conserved salt bridges, whereas in RnPepT2ECD only one salt bridge is observed. Small-angle X-ray diffraction experiments indicated that the extra-cellular domains form a compact structural arrangement, although the lobe conformation of PepT2ECD was more dynamic than PepT1. Using the X-ray crystal structures of the M. musculus and R. norvegicus ECDs, and the trans-membrane domain of PepTso from Shewanella oneidensis, the first structure-based homology models of H. sapiens PepT1 and PepT2 were constructed. The hybrid models indicated that the ECD sits on top of the transporters. Two-electrode voltage clamp studies then revealed that the ECDs do not play a part in the transport mechanism of the transporter, although PepT1ECD may play a role in transporter stability. Surface plasmon resonance binding assays were performed between the ECDs and the intestinal proteases trypsin and α-chymotrypsin. An interaction between both ECDs and α-chymotrypsin was observed, although this interaction could not be saturated using this assay. Trypsin binding however, could be saturated for both MmPepT1ECD and RnPepT2ECD giving Kds of 90 ± 20 and 170 ± 30 μM. Physiologically the interaction would give trypsin a predisposition for the peptide transporter ECD; locating the protease in the vicinity of the transporter and aiding the presentation of substrate. The interaction between trypsin and PepT1ECD was explored further with a mutational analysis of potential binding residues. The results could not locate the binding site definitively, however, the work did highlight the probable binding face which is perhaps conserved between PepT1 and PepT2 ECD
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
Biochemical and biophysical studies of the prokaryotic proton dependent oligopeptide transporters
Full text linkThe proton dependent oligopeptide transporters (POT family) are members of the Major Facilitator Superfamily of secondary active transporter proteins. They use the transmembrane proton gradient to drive the uptake of di- and tripeptides into the cytoplasm. Members of the family are highly conserved in pro- and eukaryotic genomes, and in humans they are responsible for the oral absorption of many drug families, including β-lactam antibiotics. Recently, the crystal structures of PepTSo and PepTSt, two prokaryotic homologues of the human proteins PepT1 and PepT2, captured the proteins in two distinct conformations, providing insight into the structural aspects of the transport mechanism. A protocol was designed for functional liposome reconstitution of POT proteins, and transport assays were conducted to characterise their substrate specificity, pH dependence and kinetic properties. Using site-directed mutagenesis, we identified binding site residues involved in peptide recognition and proton translocation, and distinguished between the two roles by comparing protein activity in proton- and peptide-driven conditions. We also investigated the roles of key residues in the conformational transitions that accompany the transport cycle, using data from biochemical assays, molecular dynamics simulations and modeling, as well as electron paramagnetic resonance measurements. In addition, several bacterial POT members were screened for crystallisation, in order to assess their stability and crystal diffraction quality in different detergents. Further work was performed with bacterial POT homologues YdgR and GkPOT, including binding studies using NMR spectroscopy and assaying drug transport in vivo and in vitro. Together, the data establish bacterial POTs as model systems for studying the mammalian oligopeptide transporters, and a mechanistic model for peptide transport is proposed
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
Structural and biophysical characterisation of marine viruses and amino acid transporters
No full textThe field of structural biology enables us to study the fundamental architecture of
biological samples in atomic detail. In this thesis, the techniques of electron cryomicroscopy
and X-ray crystallography are applied to structural and biophysical
investigations of marine viruses and membrane transporters respectively.
In part I, two marine viruses - a freshwater lake bacteriophage known as FLiP and a
helical archaeal virus known as APBV1 â are studied in order to gain insights into their
evolutionary origins. The 4Ã reconstruction of FLiP is presented, which reveals an
icosahedral capsid encapsulating an intact lipid bilayer. The presence of an internal
membrane makes FLiP unique among single-stranded DNA viruses. The major capsid
protein exhibits an upright double bâbarrel fold, which is iconic of the PRD1-adenovirus
structural lineage of double-stranded DNA viruses, indicating that FLiP should be
classified as a new member of this lineage. Secondly, the structure of APBV1 at nearatomic
resolution is reported, revealing the evolutionary adaptions undertaken for
survival in a hyperthermophilic environment. The formation of extensive hydrophobic
interfaces via the tight packing of the major virion glycoprotein contributes to the
extreme thermostability of the capsid, whilst the dsDNA genome is packaged as a lefthanded
superhelix within the capsid.
In part II, the fundamental mechanisms of membrane transport by eukaryotic amino
acid transporters is investigated. Transporters belonging to the human SLC36 family,
which play a potential role in nutrient sensing, were screened for high expression yield
and protein stability. Comprehensive screening of homologous proteins resulted in
purification of proteins belonging to the vacuolar basic amino acid transporter family of
Saccharomyces cerevisiae for crystallisation trails and transport assays. Vba4 was
successfully crystallised but the resolution obtained by X-ray diffraction prevented
structure determination. In addition, the substrate specificity and mechanism of
transport were investigated using differential scanning fluorimetry, microscale
thermophoresis and liposome-based functional assays.</p
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
Characterisation of the lytic viral peptide LysM and its target, the bacterial Lipid II flippase MurJ
No full textCell wall synthesis is a highly conserved and essential process in bacteria. The cell wall precursor, Lipid II, is transported across the plasma membrane by the flippase MurJ, a newly discovered putative antibiotic target that is non-redundant in many species. While the mechanism of flipping Lipid II was elucidated following a series of structures published during the proceedings of this DPhil project, there are still open questions about the mechanism employed by MurJ. Importantly, there are no available structures of Gram-positive MurJ, which have structural differences to their Gram-negative counterparts. The Escherichia coli-infecting levivirus, enterobacteria phage M, targets MurJ using a 37-amino acid peptide, LysM. The molecular mechanism of this natural example of MurJ inhibition has yet to be understood, and there is a need to evaluate the potential of viral inhibitors from the perspective of antibiotic discovery. This thesis combines in silico, in vivo, and in vitro experiments for the characterisation of LysM, determining that it is a helical peptide that spans the membrane with an amphipathic C-terminal helix that lies on the membrane surface. A helical kink occurs at the essential residue D18, orienting the C-terminal and N-terminal helices near-perpendicular to each other, with the N-terminal helix pointing straight down into the membrane. One face of the peptide confers lytic activity through a concerted contribution of polar and hydrophobic residues, which reveals a putative binding face between LysM and MurJ. A purification strategy for LysM was also established that provides a platform for future binding and structural studies, and constructs were made for confirming the N-in topology and for the co-expression of LysM and MurJ. B. subtilis MurJ was purified in multiple conditions and structurally screened. However, stability and binding studies suggested that this protein was unsuitable for further study. Homologues were screened, along with the novel flippase Amj, identifying suitable constructs for future purification and structural studies. These experiments and constructs allow for a wealth of future characterisation and establish a direct pathway for co-purification of the putative complex for Cryo-EM
Understanding the role of transporters and ion channels in lysosomal homeostasis
Full text linkCellular activity is regulated based on a range of cues to maintain homeostasis. The lysosome is a key regulator of cellular activity as it acts as a signaling hub for metabolic regulation and is the main site of biomolecule degradation. Following the degradation of biomolecules, including proteins, glycans, nucleic acids and lipids, nutrients are exported out of the lysosome by efflux transporters, which provide nutrients for cellular anabolic activity. Efflux transporters maintain lysosomal homeostasis by preventing the deleterious accumulation of nutrients within the lumen. Here, the mechanism of cystine transport at the lysosomal membrane by cystinosin is characterized using two-electrode voltage clamp. This work provides novel insights into the mechanisms that maintain nutrient homeostasis in lysosomes, and how they are disrupted in the lysosomal storage disorder cystinosis. Lysosomal homeostasis is also preserved by protein signaling, which regulates lysosome biogenesis, trafficking, fusion, and activity. A key regulator of cell signaling at the lysosomal membrane is the ion channel TRPML1. Numerous functions have been associated with TRPML1 activity, but its mechanisms of action remain poorly understood. This is in part due to the lack of specific tools to investigate its functions (modulators) and localization (antibodies). Nanobodies have previously been used as tools to investigate the mechanisms and localization of membrane proteins in cells. Here, high-affinity TRPML1 synthetic nanobodies (sybodies) were generated and characterized biophysically and structurally. A TRPML1-specific bivalent construct was engineered and applied to STochastic Optical Reconstruction Microscopy (STORM) imaging and immunoprecipitation of endogenous TRPML1. Future work will aim to use these binders and protocols to investigate the role of TRPML1 in neurodegeneration, which can result from the loss of lysosomal homeostasis. Moreover, the protocols described here will enable to engineer sybodies against additional lysosomal targets to investigate their role in lysosomal homeostasis
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