Peptidoglycan glycosyltransferases (PGTs) are highly conserved bacterial enzymes that catalyze the polymerization of the lipidic disaccharide, Lipid II, to form individual peptidoglycan (PG) strands which are subsequently cross-linked to form mature PG, the major skeletal component of the bacterial cell wall. Recent advances in the preparation of well-defined PGT substrates have enabled the biochemical characterization of Lipid II polymerization by the PGTs. In the course of these studies, we have observed that a distinctive lag phase in the initial rate of PG synthesis by the PGTs can be abrogated if the enzyme is preincubated with Lipid IV, the shortest PG fragment. The origins of this lag phase are intriguing because the chemical transformation involved in coupling Lipid II to yield Lipid IV is identical to the transformation involved in the synthesis of longer PG fragments from Lipid II. Crystallographic structures of the PGTs with Moenomycin A, an inhibitor that is believed to bind to the same site as Lipid IV, suggest that the PGTs possess flexible regions near the putative active site that can undergo substrate-induced conformational changes to accelerate PG synthesis. However, there is currently no structural evidence on how the PGTs interact with its substrates. The work in this thesis lays the foundation for pursuing a solution structure of a Lipid IV bound PGT complex by Nuclear Magnetic Resonance (NMR) spectroscopy, enabling the study of important enzyme conformational states and structural dynamics involved in PG synthesis. Specifically, Chapter 2 of this thesis presents the biochemical evidence that the preincubation of the PGTs with a Lipid IV derivative, Gal-Lipid IV abrogates the lag phase and accelerates the initial rate of PG synthesis. Chapter 3 presents a robust methodology for obtaining multimilligram quantities of isotope labeled, monodisperse and monomeric SgtB, a PGT from a clinically relevant pathogen, Staphylococcus aureus for solution structural studies. Chapter 4 describes the systematic development of a methodology for producing a well-behaved, stable sample of Moenomycin A bound SgtB for NMR spectroscopy. Chapter 5 delineates the adaptation of the methodology described in Chapter 4 for pursuing the solution structure of Lipid IV bound SgtB. / Chemistry and Chemical Biology

NMR crystallography is a new and developing area. Unlike solution state NMR, solid state NMR has the potential for structural studies of large, motionally restricted biological macromolecules, such as proteins in crystals which may, or may not, diffract. However, finding the best and the most useful sample form and geometry is still a major obstacle to rapid progress. Little has been reported about protein sample preparation for any class of protein for NMR crystallography, mainly since the availability of NMR labelled proteins is still not routine, especially for eukaryotic membrane proteins. The amino acid L-glutamate is the major excitatory neurotransmitter in the brain. Details of glutamate binding to any of its main brain or sensory receptors are not well resolved at the atomic level. In an effort to resolve the glutamate binding mechanism by solid state NMR methods, full-length taste and brain mGluR4 were expressed in E. coli, but proved to be toxic for the cells. The ligand-binding domains (LBD) of mGluR4, with various fusions for the periplasmic expression and with various fusions for expression in the cytoplasm therefore, were used. Solubilisation and then purification of the LBD from inclusion bodies is still under way, no crystals of mGluR4 for NMR were, therefore, grown. Initial NMR spectra of labelled 13C, 15N and 17O glutamate have been recorded to verify sensitivity requirements. Using homology modelling, a model for the truncated version of the ligand binding domain of mGluR4 has been constructed as a basis for designing solid state NMR experiments to probe the ligand binding site in the receptor. Bacteriorhodopsin is a large membrane protein and a model for G-protein coupled receptors (GPCRs). Spectra of bacteriorhodopsin produced in H. salinarium in purple membrane are reported here and compared to spectra of the protein crystallised from bicelles. Optimal conditions for producing spectra suitable for spectral assignment are reported as an initial step towards spectral resolution. Three differently labelled samples of bacteriorhodopsin were prepared to test the applicability of the various assignment strategies and the effects of deuteration on quality of solid state NMR spectra of a large, crystalline membrane protein.

The mitochondrial tyrosyl tRNA synthetases (mtTyrRS) from certain fungii are found to be bifunctional enzymes that aid in group I intron splicing in addition to charging tRNA[superscript Tyr]. This splicing activity is conferred by several insertions that are unique to these mtTyrRS. Initial biochemical evidence suggested the similar tertiary structures of the tRNA and the intron enable binding of the protein to both. However, a recently solved co-crystal structure showed that the tRNA and intron were bound on opposite faces of the protein. The intron was bound almost exclusively by a novel surface formed by several insertions in the protein. This work presents the structure of the C-terminal domain of the A. nidulans mtTyrRS (PDB ID -- 2ktl). NMR results show that the C-terminal domain contains an S4 fold with a mixed [beta]-sheet and two anti-parallel [alpha]-helices that pack against these strands. The strands [beta]1 and [beta]5 are parallel, and [beta]2 to [beta]5 are arranged anti-parallel to each other. The C-terminal domain from A. nidulans mtTyrRS has three insertions in its sequence that make it almost twice the size of bacterial TyrRS. NMR results show that insertion 3 at the N-terminus of the domain is flexible. Insertion 4 is contained in the loop connecting [beta]2-[beta]3 and does not have a well defined structure. Insertion 5 and the C-terminal extension form two helices, [alpha]5 and [alpha]6 that fold away from the core of the protein. An extended helix ([alpha]4) between strands [beta]3 and [beta]4 was identified by NMR. Based on structural alignments with bacterial TyrRS, this helix was classified as a novel insertion 4b in the C-terminal domain. Conserved positively charged residues used to bind the tRNA are found in the turn between the anti-parallel [alpha]-helices and the turn connecting strands [beta]4-[beta]5. Based on a comparison with other TyrRS structures, the three insertions are positioned away from the tRNA binding site. The insertions form a novel RNA binding surface that could interact with the intron. Since these insertions are found in loop and termini regions, they could be a structural adaptation acquired by these splicing mtTyrRS. NMR spectra of the full length TyrRS from B. stearothermophilus and mtTyrRS from A. nidulans indicate that the motion of the C-terminal domain is coupled to that of the full length protein. This provides new information regarding the organization of the full length TyrRS. / text

Η αποικοδόμηση των ενδοκυτταρικών πρωτεϊνών μέσω του μονοπατιού ουβικιτίνης-πρωτεοσώματος αποτελεί βασική διαδικασία που εξυπηρετεί σημαντικές ομοιοστατικές λειτουργίες του κυττάρου. Η ουβικιτίνη δεσμεύεται ομοιοπολικά στις πρωτεΐνες-στόχους μέσω ενός καταρράκτη ενζυμικών αντιδράσεων, στον οποίο καθοριστικό ρόλο παίζουν τα Ε3 ένζυμα, οι Ε3 λιγάσες ουβικιτίνης. Η πρωτεΐνη Arkadia είναι μια Ε3 λιγάση με έναν χαρακτηριστικό RING τομέα 69 αμινοξικών καταλοίπων στο C-τελικό άκρο της, μέσω του οποίου ασκεί την δράση της. Εμπλέκεται στο TGF-β σηματοδοτικό μονοπάτι το οποίο ρυθμίζει θετικά, ουβικιτινιλιώνοντας και στοχεύοντας για αποικοδόμηση πρωτεΐνες που αποτελούν αρνητικούς ρυθμιστές του. Κύριο χαρακτηριστικό του RING τομέα της Arkadia, ο οποίος έχει μελετηθεί και χαρακτηριστεί δομικά μέσω πολυπυρηνικής και πολυδιάστατης NMR φασματοσκοπίας, είναι η δέσμευση δύο ιόντων Zn2+ με χαρακτηριστικό διασταυρώμενο τρόπο (cross brace). Γνωρίζοντας τον πολύ σημαντικό ρόλο του Zn στην δομή και κατ’επέκταση στην δραστικότητα των RING τομέων, πραγματοποιήθηκαν μεταλλάξεις σε δύο αμινοξέα που συμμετέχουν στα μοτίβα δέσμευσης των δύο ιόντων Zn2+, οι H962C και H965C. Το μετάλλαγμα της H962C, στο οποίο εστιάζει η παρούσα εργασία, παρήχθη με βάση τις κλασσικές τεχνικές κλωνοποίησης DNA. Κατάλληλα δείγματα πρωτεΐνης εμπλουτισμένα σε πυρήνες ενεργούς στην φασματοσκοπία NMR, 15N και 13C, προετοιμάστηκαν ώστε να διεξαχθούν τα απαραίτητα πειράματα για τον προσδιορισμό της δομής. Το σύνολο των NMR φασμάτων απέδειξε ότι πρόκειται για ένα καλά δομημένο και σταθερό πολυπεπτίδιο. Τα δεδομένα από τις μετρήσεις ατομικής απορρόφησης και την επεξεργασία των φασμάτων επιβεβαίωσαν την διατήρηση της δέσμευσης δύο ιόντων Zn2+ ανά πολυπεπτίδιο, όπως στον RING τομέα του φυσικού τύπου της Arkadia. Ταυτόχρονα, πραγματοποιούνται περαιτέρω NMR μελέτες και αναλύσεις των φασμάτων με χρήση των κατάλληλων υπολογιστικών προγραμμάτων για την διερεύνηση της δομής και ικανότητας αλληλεπίδρασης με το Ε2 ένζυμο. / The degradation of the intracellular proteins through the ubiquitin-proteasome pathway is a crucial procedure that serves the cellular homeostasis. Ubiquitin is covalently attached to the target proteins through an enzymatic cascade, in which the E3 enzymes (E3 ubiquitin ligases) play a determinant role. Arkadia is an E3 ubiquitin ligase containing a characteristic RING domain in its C-terminus, comprised of 69 amino acids, which is responsible for its function. It is involved in TFG-β signaling pathway, where it ubiquitinates and subsequently leads negative regulators to proteasomal degradation. The most important characteristic of Arkadia’s RING domain, which has been studied and structurally characterized through multinuclear and multidimensional NMR spectroscopy, is that it bounds to zinc ions in a cross-brace manner. As the role of the zinc binding is critical for the structure and subsequently the activity of RING domains, His962 and His965, which participate in the zinc binding motifs, were mutated to cysteines. The Arkadia H962C mutant, on which the present work is focused, was produced through the classical techniques of DNA cloning. Protein samples were uniformly labeled in 15N and 13C nuclei in order for all the necessary NMR experiments to be carried out. The total of the NMR spectra demonstrated that Arkadia RING mutant H962C is a well structured and stable polypeptide. Furthermore, the information that came from the atomic absorption data and the analysis of the NMR spectra confirmed that the RING mutant maintains the RING wild type ability to bind two zinc ions. At the same time, further NMR studies are being carried out in order to investigate its structure and ability to interact with the E2 enzyme.

Οι ενεργοποιούμενοι από Ασβέστιο, μικρής αγωγιμότητας (SK), δίαυλοι καλίου παρουσιάζουν τασεο-ανεξάρτητη δράση, ενώ ενεργοποιούνται χημειομηχανικά από αυξημένα ενδοκυτταρικά επίπεδα Ασβεστίου (σε εύρος μM), όπως συμβαίνει για παράδειγμα κατά τη διάρκεια ενός δυναμικού ενέργειας. Οι SK δίαυλοι προκαλούν το φαινόμενο της μεθυπερπόλωσης (sAHP) το οποίο περιορίζει την συχνότητα πυροδότησης του νευρώνα κατά τη διάρκεια ενός δυναμικού ενέργειας. Η υψηλή συγγένεια δέσμευσης του Ασβεστίου στους SK διαύλους οφείλεται στην ανεξάρτητη Ασβεστίου αλληλεπίδραση της SK α-υπομονάδας με το C-τελικό τμήμα της Καλμοδουλίνης (TR2C), ιδιότητα που χαρακτηρίζει τους SK διαύλους στην μεγάλη οικογένεια των διαύλων καλίου. Το TR2C τμήμα δεσμεύεται σε μια συγκεκριμένη περιοχή της α-υπομονάδας των SK διαύλων η οποία ονομάζεται CaMBD (CalModulin Binding Domain). Η περιοχή αυτή εντοπίζεται ενδοκυτταρικά στο ελικοειδές C-τελικό τμήμα της συγκεκριμένης υπομονάδας. Σε μια προσπάθεια μελέτης της αλληλεπίδρασης της ελεύθερης Ασβεστίου Καλμοδουλίνης και της CaMBD περιοχής, εφαρμόσαμε φασματοσκοπία NMR σε ένα χαρακτηριστικό της αλληλεπίδρασης σύμπλοκο, που αποτελούνταν από το C-τελικό τμήμα της Καλμοδουλίνης (TR2C) και ένα συνθετικό πεπτίδιο 23 αμινοξέων, αντιπροσωπευτικό της CaMBD περιοχής. Οι πειραματικές συνθήκες ρυθμίστηκαν με χρήση τεχνικών φθορισμομετρίας προκειμένου να καθοριστεί το βέλτιστο περιβάλλον για την αλληλεπίδραση. Η δομή της απο-Καλμοδουλίνης, σε αλληλεπίδραση με τo CaMBD τμήμα, μελετήθηκε με χρήση υψηλής ανάλυσης φασματοσκοπίας NMR (600 & 900 MHz) μέσω 2D και 3D πολυπηρυνικών (1Η, 13C, 15N) φασμάτων. Η χαρτογράφηση της διαταραχής των χημικών μετατοπίσεων στο υπό αλληλεπίδραση TR2C τμήμα αποκαλύπτει την επιφάνεια αλληλεπίδρασης μεταξύ TR2C και CaMBD τμήματος ενώ η αναλυτική δομική μελέτη οδηγεί σε μια NMR δομή για το TR2C τμήμα στο σύμπλοκο με το CaMBD τμήμα. / Small conductance Ca2+-activated potassium (SK) channels are voltage independent, chemo-mechanically gated K+ channels, which utilize as their gating cue increases in the levels of intracellular Ca2+ such as occur during an action potential. SK channels underlie the slow afterhyperpolarization (sAHP) that limits the firing frequency during an action potential. SK channels are gated solely by intracellular Ca2+ in the submicromolar range. This high affinity for Ca2+ results from Ca2+-independent association of the SK α-subunit with calmodulin C-terminal domain (TR2C), a property unique among the large family of potassium channels. TR2C binds to a specific region of the α-subunit of SK channels called the CaM binding domain (CaMBD), located intracellular and immediately C-terminal to the inner helix. In an attempt to elucidate the interaction between apo-CaM and CaMBD we apply NMR Spectroscopy to a minimal complex consisting of the C-terminal domain of CaM and a synthetic 23-residue peptide corresponding to the CaMBD core region. Experimental conditions were fine-tuned through Fluorescence spectroscopy in order to identify the optimum conditions for this interaction. The structure of apo-CaM, complexed with the CaMBD, was studied by high analysis NMR spectroscopy (600 & 900 MHz) through 2D and 3D heteronuclear (1Η, 13C, 15N) spectra. Chemical shift variation mapped over the complexed apo-CaM with sk2 peptide reveals the interaction interface while the detailed conformational analysis results to an NMR solution structure.

The mathematical modeling and simulation of the flow of fluid through porous media are important in many areas. Relative permeability and capillary pressure functions are macroscopic properties that are defined within the mathematic model. Accurate determinations of these functions are of great importance. An established inverse methodology provides the most accurate estimates of the unknown functions from the available data. When the inverse method is used to determine the flow functions, the media properties, absolute permeability and porosity are typically represented by single average values for the entire sample. Fortunately, an advanced core analysis tools utilizing nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI) to determine complete distributions of porosity and permeability has been developed. The process for determining multiphase properties from experimental data is implemented with the computer program SENDRA. This program is built around a two-dimension, two-phase simulator. In this thesis, the computer code is extended to represent all three spatial coordinate directions so that the porosity and permeability distributions in three-dimensional space can be taken into account. Taking the sample's heterogeneity into account is expected to obtain more accurate multiphase property. Three synthetic experiments are used to show the erroneous estimation of flow functions associated with the homogeneity assumption. A proposal approach is used to predict the relative permeability of wetting phase using NMR relaxation data. Several sets of three-dimensional NMR experiments are performed. Three-dimensional saturation distribution and relaxation are determined. Relative permeability of wetting phase are calculated by applying an empirical relation. This approach provides a in situ measurement of relative permeability of wetting phase from NMR data.

Calmodulin (CaM) is a ubiquitous calcium sensor protein which binds and activates a variety of enzymes involved in cell signaling pathways. In its calcium loaded state, CaM is extremely resistant to heat denaturation, with a melting temperature (Tm) of around 115°C. In this study, Xenopus laevis CaM was prepared such that the eight phenylalanine residues were substituted with 3-fluorophenylalanine. 19F NMR studies then focused on properties of the hydrophobic core associated with the folding process at temperatures near the regime where the protein is completely folded. Near 70°C, near-UV circular dichroism and 1H NMR-based measurements of protein diffusion rates reveal the onset of a stable, expanded near-native folding intermediate. 19F NMR solvent isotope shifts reveal a gradual loss of water from the hydrophobic core with increasing temperature, until the point at which the near-native intermediate state is attained. At this point, water is observed to enter the hydrophobic core and destabilize the protein. Paramagnetic shifts from dissolved oxygen reveal an increase in oxygen accessibility with temperature until the near-native intermediate is reached, whereupon oxygen solubility decreases. Taken together, we conclude that hydrophobicity of the protein interior increases with temperature, until a dry near-native state is established, whereupon water cooperatively enters and destabilizes the hydrophobic core. 19F CPMG experiments provide a measure of the interconversion between the folded state and the dry near-native intermediate; at higher temperatures, folding rates are on the order of 10,000 Hz. Moreover, as temperature is lowered, folding rates increase, presumably because the effect of off-pathway misfolding events on the exchange process is diminished.

Calmodulin (CaM) is a ubiquitous calcium sensor protein which binds and activates a variety of enzymes involved in cell signaling pathways. In its calcium loaded state, CaM is extremely resistant to heat denaturation, with a melting temperature (Tm) of around 115°C. In this study, Xenopus laevis CaM was prepared such that the eight phenylalanine residues were substituted with 3-fluorophenylalanine. 19F NMR studies then focused on properties of the hydrophobic core associated with the folding process at temperatures near the regime where the protein is completely folded. Near 70°C, near-UV circular dichroism and 1H NMR-based measurements of protein diffusion rates reveal the onset of a stable, expanded near-native folding intermediate. 19F NMR solvent isotope shifts reveal a gradual loss of water from the hydrophobic core with increasing temperature, until the point at which the near-native intermediate state is attained. At this point, water is observed to enter the hydrophobic core and destabilize the protein. Paramagnetic shifts from dissolved oxygen reveal an increase in oxygen accessibility with temperature until the near-native intermediate is reached, whereupon oxygen solubility decreases. Taken together, we conclude that hydrophobicity of the protein interior increases with temperature, until a dry near-native state is established, whereupon water cooperatively enters and destabilizes the hydrophobic core. 19F CPMG experiments provide a measure of the interconversion between the folded state and the dry near-native intermediate; at higher temperatures, folding rates are on the order of 10,000 Hz. Moreover, as temperature is lowered, folding rates increase, presumably because the effect of off-pathway misfolding events on the exchange process is diminished.

Protein folding pathways can be extraordinarily complex. In this study, circular dichroism (CD) and 19F NMR are used to investigate the folding network of calmodulin, a calcium-binding protein, which is biosynthetically enriched with 3-fluorophenylalanine. In calmodulin’s calcium-loaded state, CD experiments identify the existence of a folding intermediate along a heat-denaturation pathway. In comparison to the native state, 19F NMR solvent isotope shifts reveal decreased accessibility of water to hydrophobic core, whereas O2 paramagnetic shifts show increased hydrophobicity of this folding intermediate. 15N-1H and methyl 13C-1H HSQC NMR spectra demonstrate that this folding intermediate retains a near-native tertiary structure, whose hydrophobic interior is highly dynamic. 19F NMR CPMG relaxation dispersion measurements suggest that this near-native intermediate state is transiently adopted below the temperature associated with its onset. The folding network also involves an unproductive off-pathway intermediate. In contrast, calmodulin’s calcium-free state exhibits a simpler folding process which lacks discernible intermediates.

In this thesis, we document a comprehensive study of the cation-directed self-assembly of three guanosine derivatives: i) guanosine 5'-monophosphate (5'-GMP), ii) guanosine 5'-thiomonophosphate (5'-GSMP), and iii) 2',3',5'-O-triacetylguanosine (TAG). We discovered that, under the neutral pH condition, Na2(5'-GMP) molecules self-assemble into a right-handed helix structure consisting of alternating all-C2'endo and all-C3'endo planar G-quartets stacking on top of each other with a 30° twist. This self-assembled supramolecular structure uses multiple non-covalent forces (e.g., hydrogen-bond, phosphate-hydroxyl, pi-pi (base-base) stacking, ion-carbonyl, and ion-phosphate) to align individual monomers in a way that resembles RNA and DNA sequences in which covalent bonds are used to link monomers. Na+ ions are located in the channel and surface sites of the G-quadruplex. In contrast, under acidic pH conditions, Na2(5'-GMP) molecules self-assemble into a continuous right-handed helix where guanine bases are hydrogen-bonded in a lock-washer fashion with only C3'-endo monomers. Na+ ions are absent in the channel site due to smaller channel radius and lesser repulsions between phosphate groups (-1 vs. -2 charge under neutral pH) contribute to the stronger stacking mechanism. In Na2(5'-GSMP), a longer phosphate bond compared with Na2(5'-GMP) allows stronger P-O-…Na+…-O-P interactions to occur, thus enhancing self-assembly. Solid-state NMR, FT-IR, powder x-ray diffraction, model building, and calculation showed that Na2(5'-GSMP) forms the same self-assembled structure as Na2(5'-GMP) but with significantly greater tendency. This study proves that single-bond modification can enhance stacking in G self-assemblies, and shows direct evidence that Na+ ions reside at the surface (phosphate) sites. Lastly, using lipophilic TAG, we were able to show for the first time that trivalent lanthanide metal ions can facilitate G-quartet formation. A new mode of metal ion binding in G-quartet structures (i.e., a triple-decker G dodecamer containing a single metal ion in the central G-quartet) is reported. We also report the first 1H and 43Ca NMR characterization of Ca-templated G-quartet formation in a [TAG8-Ca]2+ octamer. / Thesis (Ph.D, Chemistry) -- Queen's University, 2012-06-27 16:53:43.359