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1
Andres, Dorothee. "Biophysical chemistry of lipopolysaccharide specific bacteriophages." Phd thesis, Universität Potsdam, 2012. http://opus.kobv.de/ubp/volltexte/2012/5926/.
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Carbohydrate recognition is a ubiquitous principle underlying many fundamental biological processes like fertilization, embryogenesis and viral infections. But how carbohydrate specificity and affinity induce a molecular event is not well understood. One of these examples is bacteriophage P22 that binds and infects three distinct Salmonella enterica (S.) hosts. It recognizes and depolymerizes repetitive carbohydrate structures of O antigen in its host´s outer membrane lipopolysaccharide molecule. This is mediated by tailspikes, mainly β helical appendages on phage P22 short non contractile tail apparatus (podovirus).
The O antigen of all three Salmonella enterica hosts is built from tetrasaccharide repeating units consisting of an identical main chain with a distinguished 3,6 dideoxyhexose substituent that is crucial for P22 tailspike recognition: tyvelose in S. Enteritidis, abequose in S. Typhimurium and paratose in S. Paratyphi. In the first study the complexes of P22 tailspike with its host’s O antigen octasaccharide were characterized. S. Paratyphi octasaccharide binds less tightly (ΔΔG≈7 kJ/mol) to the tailspike than the other two hosts. Crystal structure analysis of P22 tailspike co crystallized with S. Paratyphi octasaccharides revealed different interactions than those observed before in tailspike complexes with S. Enteritidis and S. Typhimurium octasaccharides. These different interactions occur due to a structural rearrangement in the S. Paratyphi octasaccharide. It results in an unfavorable glycosidic bond Φ/Ψ angle combination that also had occurred when the S. Paratyphi octasaccharide conformation was analyzed in an aprotic environment. Contributions of individual protein surface contacts to binding affinity were analyzed showing that conserved structural waters mediate specific recognition of all three different Salmonella host O antigens.
Although different O antigen structures possess distinct binding behavior on the tailspike surface, all are recognized and infected by phage P22. Hence, in a second study, binding measurements revealed that multivalent O antigen was able to bind with high avidity to P22 tailspike. Dissociation rates of the polymer were three times slower than for an octasaccharide fragment pointing towards high affinity for O antigen polysaccharide. Furthermore, when phage P22 was incubated with lipopolysaccharide aggregates before plating on S. Typhimurium cells, P22 infectivity became significantly reduced.
Therefore, in a third study, the function of carbohydrate recognition on the infection process was characterized. It was shown that large S. Typhimurium lipopolysaccharide aggregates triggered DNA release from the phage capsid in vitro. This provides evidence that phage P22 does not use a second receptor on the Salmonella surface for infection. P22 tailspike binding and cleavage activity modulate DNA egress from the phage capsid. DNA release occurred more slowly when the phage possessed mutant tailspikes with less hydrolytic activity and was not induced if lipopolysaccharides contained tailspike shortened O antigen polymer. Furthermore, the onset of DNA release was delayed by tailspikes with reduced binding affinity. The results suggest a model for P22 infection induced by carbohydrate recognition: tailspikes position the phage on Salmonella enterica and their hydrolytic activity forces a central structural protein of the phage assembly, the plug protein, onto the host´s membrane surface. Upon membrane contact, a conformational change has to occur in the assembly to eject DNA and pilot proteins from the phage to establish infection.
Earlier studies had investigated DNA ejection in vitro solely for viruses with long non contractile tails (siphovirus) recognizing protein receptors. Podovirus P22 in this work was therefore the first example for a short tailed phage with an LPS recognition organelle that can trigger DNA ejection in vitro. However, O antigen binding and cleaving tailspikes are widely distributed in the phage biosphere, for example in siphovirus 9NA. Crystal structure analysis of 9NA tailspike revealed a complete similar fold to P22 tailspike although they only share 36 % sequence identity. Moreover, 9NA tailspike possesses similar enzyme activity towards S. Typhimurium O antigen within conserved amino acids. These are responsible for a DNA ejection process from siphovirus 9NA triggered by lipopolysaccharide aggregates. 9NA expelled its DNA 30 times faster than podovirus P22 although the associated conformational change is controlled with a similar high activation barrier. The difference in DNA ejection velocity mirrors different tail morphologies and their efficiency to translate a carbohydrate recognition signal into action.Kohlenhydraterkennung ist ein fundamentales Prinzip vieler biologischer Prozesse wie z.B. Befruchtung, Embryogenese und virale Infektionen. Wie aber Kohlenhydratspezifität und –affinität in ein molekulares Ereignis übersetzt werden, ist nicht genau verstanden. Ein Beispiel für ein solches Ereignis ist die Infektion des Bakteriophage P22, der drei verschiedene Salmonella enterica (S.) Wirte besitzt. Er erkennt und depolymerisiert die repetitiven Einheiten des O Antigens im Lipopolysaccharid, das sich in der äußeren Membran seines Wirtes befindet. Dieser Schritt wird durch die Tailspikes vermittelt, β helicale Bestandteile des kurzen, nicht kontraktilen Schwanzapparates von P22 (Podovirus). Das O Antigen aller drei Salmonella enterica Wirte besteht aus sich wiederholenden Tetrasacchariden. Sie enthalten die gleiche Hauptkette aber eine spezifische 3,6 Didesoxyhexose Seitenkette, die für die P22 Tailspikeerkennung essentiell ist: Tyvelose in S. Enteritidis, Abequose in S. Typhimurium und Paratose in S. Paratyphi. Im ersten Teil der Arbeit wurde die Komplexbildung von P22 Tailspike mit O Antigen Octasaccharidfragmenten der drei verschiedenen Wirte untersucht. S. Paratyphi Octasaccharide binden mit einer geringeren Affinität (ΔΔG≈7 kJ/mol) an den Tailspike als die beiden anderen Wirte. Die Kristallstrukturanalyse des S. Paratyphi Octasaccharides komplexiert mit P22 Tailspike offenbarten unterschiedliche Interkationen als vorher mit S. Enteritidis und S. Typhimurium Oktasaccharidkomplexen mit Tailspike beobachtet wurden. Diese unterschiedlichen Interaktionen beruhen auf einer strukturellen Änderung in den Φ/Ψ Winkeln der glykosidischen Bindung. Die Beiträge von verschiedenen Proteinoberflächenkontakten zur Affnität wurden untersucht und zeigten, dass konservierte Wasser in der Struktur die spezifische Erkennung aller drei Salmonella Wirte vermittelt. Obwohl die verschiedenen O Antigen Strukturen unterschiedliches Bindungsverhalten auf der Tailspikeoberfläche zeigen, werden alle vom Phagen P22 erkannt und infiziert. Daher wurde in einer zweiten Studie die multivalente Bindung zwischen P22 Tailspike und O Antigen charakterisiert. Die Dissoziationskonstanten des Polymers waren drei Mal langsamer als für das Oktasaccharid allein, was auf eine hohe Affinität des O Antigens schließen lässt. Zusätzlich wurde gezeigt, dass die Aggregate des Lipopolysaccharids in der Lage sind, die Infektiösität vom P22 Phagen zu reduzieren. Ausgehend davon wurde in einer dritten Studie die Bedeutung der Kohlenhydrat Erkennung auf den Infektionsprozess untersucht. Große S. Typhimurium Lipopolysaccharide Aggregate bewirkten die DNA Freisetzung vom P22 Kapsid. Dies deutet darauf, dass der P22 Phage keinen weiteren Rezeptor für die Infektion auf der Oberflächen seines Wirtes verwendet. Zusätzlich moduliert die P22 Tailspike Aktivität den Ausstoss der DNA vom P22 Phagen: Er ist langsamer, wenn der Phage Tailspikes besitzt, die weniger hydrolytisch aktiv sind und wurde nicht induziert, wenn Lipopolysaccharid eingesetzt wurde, dass zuvor mit Tailspike hydrolysiert wurde. Darüber hinaus wurde der Start der DNA Ejektion verzögert, wenn Tailspikes mit verminderter Affinität am Phagen vorhanden waren. Die Ergebnisse führten zu einem Modell für die Infektion von P22: Tailspikes positionieren den Phagen auf Salmonella enterica und ihre Aktivität drückt ein zentrales Strukturprotein des Phagen, das Stöpselprotein, auf die Membranoberfläche. Aufgrund des Membrankontaktes findet eine Konformationsänderung statt die zur Ejektion der Pilotproteine und zur Infektion führt. Vorhergehende Studien haben bisher nur die DNA Ejektion in vitro für Viren mit langen, nicht kontraktilen Schwänzen (Siphoviren) mit Proteinrezeptoren untersucht. In dieser Arbeit wurde das erste Mal die DNA Ejektion für einen Podovirus mit LPS Erkennung in vitro gezeigt. Die O Antigen Erkennung und Spaltung durch Tailspikeproteine gibt es häufig in der Phagenbiosphere, z.B. am Siphovirus 9NA. Die Kristallstrukturanalyse von 9NA Tailspike zeigt eine komplett gleiche Struktur, obwohl beide Proteine nur zu 36% Sequenzidentität besitzen. Zusätzlich hat 9NA Tailspike ähnliche enzymatische Eigenschaften. Diese ist für den DNA Ejektionsprozess im Siphovirus 9NA verantwortlich, der auch durch LPS Agreggate induziert wird. 9NA stößt dabei seine DNA 30 Mal schneller aus als Podovirus P22 obwohl die damit verbundene Konformationsänderung mit einer ähnlich hohen Aktivierungsbarriere kontrolliert wird. Daher spiegeln die Unterschiede in der DNA Ejektionsgeschwindigkeit der verschiedenen Tailmorphologien die Effezienz wieder, mit der die spezifische Kohlenhydraterkennung in ein Signal umgewandelt wird.
2
Cuccia, Louis A. "Biophysical properties of dimeric phospholipids." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=42007.
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A series of unusual bipolar and bis-phospholipids (dimeric phospholipids) have been studied. The structure, conformation, morphology and biophysical properties of the resulting phospholipid aggregates were investigated.Deuterium magnetic resonance spectroscopy ($ sp2$H NMR) was used to study and characterize the conformation and acyl chain order in oriented bipolar lipid membranes. The $ sp2$H-NMR studies indicated a large and constant value for the order parameter (S$ rm sb{mol})$ for all positions along the bipolar lipid diacyl chain for mechanically oriented, magnetically oriented and unoriented samples. This indicates that the great majority ($>$90%) of the bipolar lipid exists in a highly ordered spanning conformation.
Dimeric phospholipid aggregate morphologies were studied using $ sp{31}$P NMR, small angle X-ray scattering, electron microscopy, differential scanning calorimetry, and the Langmuir film balance technique in order to study the relationship between lipid structure and aggregate morphology. Dimeric phospholipids favour a lamellar morphology. A number of lipid structure-dependent features have been observed including tri-lamellar structures, extended ripple phases and hexagonal phases.
Dimeric and non-hydrolyzable phospholipids were used to study the phenomenon of interfacial activation of extracellular phospholipase A$ sb2$ (EC. 3.1.1.4) (PLA$ sb2)$ in relation to lipid phase, substrate conformation and mobility. Kinetic results and product analyses are consistent with a situation where the spanning conformer of bipolar phospholipids is resistant to PLA$ sb2$-catalyzed hydrolysis but the hairpin conformer is readily hydrolyzed. Finally, an analysis of interfacial kinetics in non-hydrolyzable matrices indicated varying degrees of interfacial inhibition and hydrolysis product activation. This has not been explicitly recognized before and affects the choice of assay conditions for PLA$ sb2.$
3
Wisner, Daniel A. "Biophysical studies of biological phosphates /." The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu148732651171337.
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Danial, John Shokri Hanna. "Imaging lipid phase separation in droplet interface bilayers." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:34bb015f-2bc1-43bb-bc29-850e0b55edac.
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The spatiotemporal organization of membrane proteins is implicated in cellular trafficking, signalling and reception. It was proposed that biological membranes partition into lipid rafts that can promote and control the organization of membrane proteins to localize the mentioned processes. Lipid rafts are thought to be transient (microseconds) and small (nanometers), rendering their detection a challenging task. To circumvent this problem, multi-component artificial membrane systems are deployed to study the segregation of lipids at longer time and length scales. In this thesis, multi-component Droplet Interface Bilayers (DIBs) were imaged using fluorescence and interferometric scattering microscopy. DIBs were used to examine and manipulate microscopic lipid domains and to observe, for the first time, transient nanoscopic lipid domains. The techniques and results described here will have important implications on future research in this field.
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Zimanyi, Eric Norman. "Theoretical advances toward understanding recent experiments in biophysical chemistry." Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/73181.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2012.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
Several theoretical advances are presented, with the common theme of helping better understand and guide recent experiments in biophysical chemistry. In Chapter 2, I consider a recent criticism of the Jarzynski equality, notably that a breakdown in the connection between work and changes in the Hamiltonian for time-dependent systems causes the Jarzynski equality to produce unphysical results. I discuss the relationship between two possible definitions of free energy and demonstrate that it is indeed possible to obtain physically relevant free energy profiles from the Jarzynski equality, thereby resolving the recent questions in the literature. Next, I consider several aspects of coherent resonance energy transfer. In Chapter 3, I present a theory for coherent resonance energy transfer based on classical electrodynamics and demonstrate how it is able to capture dynamics in the coherent regime, the incoherent regime, and in between these two limits. In Chapter 4, I present a quantum theory for resonant energy transfer based on using a variational polaron transform to optimally split the Hamiltonian into a zeroth-order part and a perturbation. I then apply a quantum master equation to obtain the dynamics of energy transfer for various parameters. Finally, in Chapter 5, I examine whether it is possible to use the known exact equilibrium state of the system to improve the variational procedure.
by Eric Norman Zimanyi.
Ph.D.
6
Damianoglou, Angeliki. "Biophysical characterisation of peptides and proteins." Thesis, University of Warwick, 2010. http://wrap.warwick.ac.uk/3664/.
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Battle, Michele Marie. "Biophysical studies of phospholipid systems." Thesis, University of Greenwich, 1992. http://gala.gre.ac.uk/6109/.
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Isaksson, Mikael. "On the quantitative analysis of electronic energy transfer/migration in proteins studied by fluorescence spectroscopy." Doctoral thesis, Umeå : Department of Chemistry, Umeå University, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1009.
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Andres, Dorothee Verfasser], and Robert [Akademischer Betreuer] [Seckler. "Biophysical chemistry of lipopolysaccharide specific bacteriophages / Dorothee Andres. Betreuer: Robert Seckler." Potsdam : Universitätsbibliothek der Universität Potsdam, 2012. http://d-nb.info/1029376824/34.
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Keatch, Steven Alexander. "Biophysical chemistry of EcoKI in physiological solutions : emulating the cell interior." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/12335.
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Production of polyamines and nucleoid-associated proteins is tightly regulated and restructures the nucleoid-associated proteins is tightly regulated and restructure the nucleoid under environmental conditions that induce DNA damage into an even more highly condensed conformation. These ‘stressful’ conditions can cause the specific methylation sequence of DNA to be lost, which leaves the DNA open to self-attack by restriction enzymes. One such enzyme is EcoKI, a type I restriction enzyme that protects the bacterial cell by destroying foreign invading DNA. Upon loss of specific methylation, EcoKI could potentially destroy the host DNA and kill the bacteria. This damaging restriction is alleviated due to partial proteolysis of EcoKI by C1pXP, although a reduced ability to destroy incoming foreign DNA is maintained. However, this method of alleviation does not exist for all type I enzymes, implying that additional restriction alleviation is required to protect bacteria. In this thesis, it has been found that the condensed structure of DNA produced by the polyamine spermidine and the nucleoid-associated protein StpA, as well as the non-specific DNA-binding of the ligand YOYO, dramatically inhibit EcoKI ATP hydrolysis an restriction activities. These results show that condensation may be a method used by bacteria to protect the nucleoid from self-attack by EcoKI under DNA-damaging conditions, and therefore forms a second mechanism of restriction alleviation. Such a condensed DNA structure may inhibit access of the enzyme to its binding site as well as inhibiting the physical ability to translocate DNA. This is in contrast to invading foreign ‘naked’ DNA in the cytoplasm, which adopts a more open conformation, and therefore forms an ideal substrate for EcoKI translocation and restriction.
11
Andres, Dorothee [Verfasser], and Robert [Akademischer Betreuer] Seckler. "Biophysical chemistry of lipopolysaccharide specific bacteriophages / Dorothee Andres. Betreuer: Robert Seckler." Potsdam : Universitätsbibliothek der Universität Potsdam, 2012. http://d-nb.info/1029376824/34.
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Massis, Sameer. "Biophysical studies and thermodynamic analysis of cathepsin B interactions." Thesis, McGill University, 1998. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21606.
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The aim of this project was to study the binding of a series of truncated forms of the propeptide to rat cathepsin B. This was done previously by using enzyme inhibition assays (Chen et al, 1996), in which binding is detectable only for those peptides that bind to the catalytic site, and hence compete with the substrate for that site. For this reason, Isothermal Titration Calorimetry (ITC) was utilised since heat detection provides a more direct measurement of binding, irrespective of its location on the enzyme. However, several difficulties were encountered which rendered this approach less feasible. Nevertheless, in the course of analysing complications of the cathepsin B system, other biophysical techniques were used in addition to ITC. In turn, this helped further characterise several aspects of cathepsin B interactions.In summary, ITC was used to study cathepsin B interactions with its propeptide, several nitrile inhibitors, leupeptin, and E64. (Abstract shortened by UMI.)
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Munshi, Sudipto. "Cloning, purification and biophysical characterization of extremophile DNA photolyases." Diss., Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/359924.
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ChemistryPh.D.
DNA photolyase (PL) is a flavoprotein that repairs UV light induced cyclobutylpyrimidine dimers (CPDs) in DNA by a photo-induced electron transfer (PET) mechanism. All our understanding about the structure and function of PL is derived from studying the mesophilic forms of the protein that have evolved to function at ambient temperature. However, the discovery of extremophiles that inhabit and thrive at thermally extreme environments on Earth has raised interesting questions about how PL functions in these organisms. Since CPD repair by PL occurs by a PET mechanism, according to Marcus theory for electron transfer, the rate constants for the ET process are expected to be exponentially sensitive to temperature, rendering the CPD repair process, as understood from studying mesophilic PLs, highly improbable at extreme temperatures. However, we have found that two extremophile PLs: a thermo-stable PL from the hyperthermophilic archaeon Sulfolobus solfataricus (SsPL) and a cold-adapted PL from the psychrophilic bacterium Colwellia psychrerythraea 34H (CpPL), are both fully competent to repair CPD-containing DNA. Thus, we hypothesize that these extremophile PLs have evolved adaptation features that are uniquely suited for function in their respective thermal environments. In order to understand how DNA repair by PL occurs at these thermal extremes, the genes for both SsPL and CpPL were cloned, the recombinant proteins were overexpressed, purified and their biophysical and DNA repair properties were characterized. Both PLs were found to contain a noncovalently bound catalytic FAD molecule, as in other PLs, but showed differences in the identity of their antenna chromophores. Unique spectral features of the cofactors, indicative of structural adaptations to their respective thermal environments were observed. CpPL was found to be extremely sensitive to its thermal and aqueous environments, whereas SsPL displayed extreme thermal stability. The formation of a unique anionic semiquinone radical state (FAD•−) was observed for the antenna cofactor-lacking mutant of SsPL, something that has never been observed in any true CPD PL characterized so far. A comparative analysis of the kinetics of temperature-dependent CPD repair by mesophilic and thermophilic PLs was performed and the results suggest the SsPL is indeed suited for CPD repair at high temperatures. Analysis of thermal denaturation and the temperature-dependent CPD repair studies also indicated the importance of the antenna cofactor in the thermophilic PLs. Finally, computational analysis of the structure of the extremophile PLs revealed unique features related to temperature adaptation. Thus, based on various experimental and computational approaches, an initial picture of how these extremophile PLs have adapted to their respective extreme thermal environments, was obtained.
Temple University--Theses
14
Nahi, Riyadh. "Synthesis and biophysical studies of nucleic acid-binding oligomers." Thesis, Cardiff University, 2016. http://orca.cf.ac.uk/97207/.
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In general, this thesis describes the design, synthesis, oligomerization and biophysical studies of novel PNA monomers. Our initial aim was to develop an efficient and inexpensive route for the synthesis of a series of novel alkyne PNA monomers bearing thymine, cytosine, adenine and guanine nucleobases suitable for Fmoc solid phase PNA synthesis strategy. These novel monomers allow functionalising the PNA sequences with alkyne functions at their Nterminus during solid phase synthesis. These novel monomers can be exploited in the click reaction applications such as a click ligation of PNA and conjugation of PNA with different substrates such as nucleic acids and proteins. As an application for ligation PNA sequences, the alkyne thymine PNA monomer was incorporated successfully into the target PNA oligomers during oligomerization. Mimicking the click (CuAAC) reaction linkers of the ligation of PNA oligomers, three novel 1,2,3-triazole functionalised building blocks were designed and prepared suitable for the Fmoc solid phase PNA synthesis strategy. Furthermore, synthesis of these building blocks specifically that is bearing thymine base represents an attempt for modification the original PNA oligomers in order to improve their drawbacks such as poor water solubility and cellular uptake or to enhance their hybridization properties. Our approach to synthesise these monomers is the Click (CuAAC) reaction which is not analogous for the routes that are used for the synthesis of the modified or unmodified PNA monomers. To investigate their effect on the biophysical properties of PNA oligomers, a single monomeric unit of these monomers was incorporated successfully into a series of mixed purine-pyrimidine 8-mer PNA sequences following the standard Fmoc-solid phase PNA synthesis conditions. In general, The Tm experiments-UV spectroscopy showed that the modified PNA oligomers containing these modified monomers still have a binding affinity with complementary sequences of DNA and RNA. The Tm values indicated that the incorporation of the 1,2,3-triazole functionalised monomers maintained or slightly reduced the thermal stability of the PNA/DNA duplexes, whereas the PNA/RNA duplexes resulted in a significantly reduced thermal stability. All of them are compared to the corresponding unmodified PNA/DNA duplexes.
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Chang, Michelle M. (Michelle Miran). "Biochemical and biophysical investigations of N-linked glycosylation pathways in archaea." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/97981.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, February 2015.Cataloged from PDF version of thesis. "December 2014."
Includes bibliographical references.
Asparagine-linked glycosylation is an abundant and complex protein modification conserved among all three domains of life. Much is known about N-glycan assembly in eukaryotes and selected bacteria, in which the oligosaccharyltransferase (OTase) carries out the en bloc transfer of glycans from polyprenyl-PP-linked donors onto asparagine side chains of acceptor proteins. The first aim of this thesis is to elucidate the biochemical details of archaeal N-linked glycosylation, specifically through in vitro analysis of the polyprenyl-P-dependent pathway of the methanogenic archaeon Methanococcus voltae. The archaeal OTase, known as AglB, utilizes a-linked dolichyl-P-trisaccharide substrate as the glycosyl donor for transfer to the acceptor protein. This dolichyl-P-glycan is generated by an initial retaining glycosyltransferase (AglK) and elaborated by additional glycosyltransferases (AglC and AgIA) to afford Dol-P-GlcNAc- Glc-2,3-diNAcA-ManNAc(6Thr)A. Despite the homology to other bacterial or eukaryotic OTases that exploit polyprenyl-PP-linked substrates, the M. voltae AglB efficiently transfers disaccharide to model peptides from the Dol-P-GlcNAc-Glc-2,3-diNAcA monophosphate. While this archaeal pathway affords the same asparagine-linked P-glycosyl amide products generated in bacteria and eukaryotes, these studies provide the first biochemical evidence revealing that despite the apparent similarities of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life. A second focus of this thesis involves biophysical studies to probe structural features and conformational dynamics of AglB. An intramolecular LRET experimental system was developed to report on substrate binding and the resulting structural transformations in AgIB. There is a strong need for detailed studies on the mechanistic and functional significance of archaeal adaptations of N-linked glycosylation, especially exploring differences between AglB and other OTases that allow AglB to utilize these unique polyprenyl-P-linked substrates. Lastly, a cell-free expression system was established for the efficient synthesis of Alg5, a yeast dolichyl-phosphate glucosyltransferase that shares high sequence similarity to AglK, the first glycosyltransferase in the M. voltae pathway. Dol-P-Glc was generated and examined to unambiguously characterize the stereochemistry of the product of Alg5.
by Michelle M. Chang.
Ph. D.
16
Mantri, Shiksha. "Engineered α-hemolysin pores with chemically and genetically-fused functional proteins." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:55450bd3-b93f-410f-b795-0110449c0da9.
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Protein engineering could be used to bring two proteins together, which don't normally interact, in an oriented configuration. Using computer modelling and experimental work involving mutagenesis, a new dimer complex, (α7)2, was engineered with two α-hemolysin (αHL) heptamers (α7) units linked via disulfide bridges in a cap-to-cap orientation. The structure of (α7)2 was confirmed by biochemical analysis, transmission electron microscopy (TEM) and single-channel electrical recording. Importantly, it was shown that the one of two transmembrane barrels of (α7)2 can insert into an attoliter liposome, while the other spans a planar lipid bilayer. (α7)2 pores spanning two bilayers were also observed by TEM. In potential, (α7)2 could be used for small molecule transfer between micron-sized vesicles (minimal cells) and would have applications in forming proto-tissues from minimal cells. Another target has been to couple a highly processive exonuclease, λ-exonuclease (λ-exo), which functions as a trimer, with the α7 pore for DNA sequencing and single molecule studies of λ-exo. Several genetic fusion constructs of λ-exo and αHL were screened and optimized for activity. By linking the N-terminus of λ-exo monomer to the C-terminus of the αHL monomer (α1), a new kind of processive exonuclease (AE) was synthesized that can form pores in bilayers. AE and wild-type α1 could be integrated into hetero-heptamers with different number of AE subunits. To achieve a hetero-heptamer with only one λ-exo trimer molecule mounted on the αHL cap, a concatemer of 2 λ-exo (exo3) was made by genetically linking the monomers of λ-exo with 15 and 17 amino acid linkers. The immediate next step is to link exo3 to α1 and then to co-assemble the exo3-α1 fusion construct with α1 to make the λ-exo-αHL pore complex. Using similar strategies as described in this thesis, other proteins could be linked to αHL increasing the scope of the nanopore technology.
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Wysoczanski, Piotr. "Structural and biophysical characterization of photoswitchable peptides and their complexes." Thesis, Cardiff University, 2012. http://orca.cf.ac.uk/44862/.
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Networks of biomacromolecular interaction form the core of internal cellular logic where a wide range of input signals get analysed and results in a genetically programmed output. This description holds not only for healthy cells but is true also for those virally infected when a virus implements new biomacromolecular elements and modifies existing networks. Those hostile interactions are crucial for the HIV virus replication cycle. Due to the scale-free nature of biomacromolecular interaction networks they are prone to malfunction if a hub (node with the highest interactions) is damaged. Perhaps the best example of such a situation can be observed in the apoptotic machinery. Overexpression, deletion or mutations (depending on the context) in apoptotic proteins can lead to various diseases, most prominently cancer. Therefore current research efforts concentrate on designing inhibitors of malfunctioning interactions. Occasionally interaction between the two binding partners relies on a well-defined secondary structures element. This allows to engineer this element and use it to bias the given interaction equilibrium towards dissociation. One such approach concentrates on α-helices that are crosslinked with azobenzene derivatives. This permits not only to increase the helical content of a peptide, which translates to increased affinity (potency), but transplants a photo-controllability. Photoswitchable peptides created in this way can be controlled externally; their affinity towards targets can be effectively photomodulated. The first chapter of the thesis concentrates on the development and evaluation of photocontrollable peptides derived from the arginine rich region of the HIV type I Rev protein. Rev, which is an early product generated from the HIV genome, binds to RRE containing mRNA and removes it from the nucleus, allowing expression of unspliced or singly spliced mRNA into different protein products. As a consequence the RRE-Rev interaction controls the infection cycle of the HIV type 1, as these later products are necessary for virus particle assembly. The α-helical peptide derived from Rev was shown to retain most of the affinity to RRE. Rev-RRE serves as a prototypical protein-RNA interaction; a model for the development of the first photo-controllable peptide that targets RNA. This research presents two approaches for the peptide production: chemical synthesis and biosynthesis (overexpression of the Rev peptide gene). Initially the water soluble azobenzene derivative was used bearing two negatively charged sulphonate substituents on the aromatic rings. Surprisingly, the first generation of the peptides had substantially decreased affinity to RRE VI as compared to the wild type Rev peptide. By applying computational chemistry techniques a possible rationalisation was found: sulphonates interact strongly with crucial arginines on the peptide making them unavailable to engage the phosphate backbone; additionally the peptide was forced into non-helical conformation prior to binding. This hypothesis was validated by crosslinking Rev peptides with azobenzene derivative deprived of the sulphonates. The second generation of Rev peptides binds RRE with high affinity and the interaction in question can be controlled effectively by light. The second chapter of the thesis focuses on the structural characterization of a photocontrollable Bak peptide bound to Bcl-xL – a prominent member of the Bcl-2 family of proteins. The Bcl-2 family of proteins includes the major regulators and effectors of the intrinsic apoptosis pathway. Cancers are frequently formed when activation of the apoptosis mechanism is compromised either by misregulated expression of prosurvival family members or, more frequently, by damage to the regulatory pathways that trigger intrinsic apoptosis. Short peptides derived from the pro-apoptotic members of the Bcl-2 family can activate mechanisms that ultimately lead to cell death. The recent development of photocontrolled peptides that are able to change their conformation and activity upon irradiation with an external light source has provided new tools to target cells for apoptosis induction with temporal and spatial control. In this thesis the first NMR solution structure of a photoswitchable peptide derived from the proapoptotic protein Bak in complex with the antiapoptotic protein Bcl-xL is reported. This structure provides insight into the molecular mechanism, by which the increased affinity of such photopeptides compared to their native forms, is achieved, and offers a rationale for the large differences in the binding affinities between the helical and nonhelical states.
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Elmlund, Hans. "Protein structure dynamics and interplay : by single-particle electron microscopy." Doctoral thesis, Stockholm : Teknik och hälsa, Technology and Health, Kungliga Tekniska högskolan, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-4669.
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McLean, Janel Renee. "Biophysical studies of anhydrous peptide structure." [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-1437.
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Deng, Xiaodi A. "Apolipoprotein A-IV Structural Determination and Biophysical Characterization." University of Cincinnati / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1368085505.
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Colegate, Rachel Marie. "A biophysical study of surfactants: an assessment of bioaccumulation and toxicity." Thesis, University of Kent, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.484072.
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Ilag, Leopold Luna. "Biochemical and biophysical aspects of molecular recognition and signalling by neurotrophins /." Stockholm, 1997. http://diss.kib.ki.se/1997/19971107ilag.
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Bachir, Alexia. "Characterization of quantum dot blinking and steric effects on fluorescence-based biophysical techniques." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=19293.
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Semiconductor nanoparticles, or quantum dots (QDs) are being increasingly applied as luminescent labels in optical studies for biophysical and cell biological applications due to their unique spectroscopic properties as compared to traditional organic fluorescent probes. However, the intermittency in the fluorescence emission of QDs, commonly referred to as 'blinking', is suggested to affect transport studies using fluctuation correlation techniques. The QD size has also been suggested to be sterically limiting for some studies. The focus of this thesis work is to examine the photophysical blinking properties and steric size effects of QD nanoparticles and determine how they influence biophysical measurements of molecular transport and protein-protein interactions. The first goal was to characterize and account for QD fluorescence blinking in transport measurements utilizing the temporal image correlation spectroscopy (TICS) technique. Fluorescence correlation techniques monitor spontaneous fluctuations in fluorescence intensity from fluorophores as they enter and exit an optically defined focal volume. Transport coefficients are recovered by fitting a calculated autocorrelation function (ACF) to appropriate analytical models that accurately represent the underlying dynamics. The QD blinking is governed by power-law statistics, which describe processes that occur on multiple time scales. As a result, QD fluorescence blinking fluctuations contribute to the calculated ACF on all times scales and can not be decoupled from diffusion fluctuations. We first present temporal image correlation spectroscopy (TICS) measurements of single QDs and ensembles of immobile QDs as well as computer simulations of blinking point emitters to deterLes nanoparticules semi-conductrices, ou particules quantiques (PQs), sont utilisées de plus en plus fréquemment comme sondes luminescentes dans les domaines de la biophysique et de la biologie cellulaire pour leurs propriétés spectroscopiques uniques et avantageuses comparées aux sondes fluorescentes organiques.Toutefois, l'intermittence de l'émission fluorescente des PQs est censée affecter les études de transport dans les techniques de corrélation de variance.Il est proposé également que les grandeurs physiques des PQs limitent également les études qui les utilisent comme sondes.L'accent du travail de cette thèse consiste à étudier les propriétés de l'intermittence photophysique et des effets des grandeurs physiques des PQ pour déterminer comment ils affectent les mesures biophysiques du transport moléculaire et des interactions protéine-protéine.Le premier objectif était de caractériser et compenser l'intermittence de la fluorescence des PQ dans les mesures des migrations utilisant la technique de spectroscopie de corrélation temporelle dans les images (TICS).Les techniques de corrélation fluorescente étudient les variations spontanées de l'intensité fluorescente de particules fluorescentes qui entrent et qui sortent d'un volume défini par un focus optique.Les coefficients de transport sont obtenus par une régression d'une fonction d'auto-corrélation (FAC) en assumant un modèle approprié qui définit le transport du système.L'intermittence des PQs est régie par une probabilité qui décrit des processus qui surviennent sur plusieurs ordres de grandeur.Donc,les fluctuations de l'intermittence des PQs ne peuvent être découplées des fluctuations venant du transport et doivent être incorp
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Pathmasiri, Wimal. "Structural and Biophysical Studies of Nucleic Acids." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8245.
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Mahajan, Sujit S. "Glycans for ricin and Shiga toxins: Synthesis and biophysical characterization." University of Cincinnati / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1307320080.
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Rosario-Jansen, Theresa. "Thiophospholipids : synthesis, biochemical studies, and biophysical properties of phospholipids chiral at phosphorus /." The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487335992902349.
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Powell, Kimberley Jade. "Synthetic and biophysical studies on the tridachiahydropyrone family of natural products." Thesis, University of Nottingham, 2014. http://eprints.nottingham.ac.uk/14228/.
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This thesis primarily details synthetic and biophysical studies on the tridachiahydropyrone family of natural products. The general aim of this work was to explore an hypothesis regarding the location and function of these metabolites, isolated from sacoglossan molluscs. Specifically, it was hypothesised that tridachiahydropyrone is synthesised photochemically from linear polyene precursors via a selective double bond isomerisation-6pi electrocyclisation sequence which occurs within the cell membrane of the producing organism. Furthermore, it was postulated that this reaction sequence, and subsequent photochemical transformations of tridachiahydropyrone into the related products phototridachiahydropyrone and oxytridachiahydropyrone, serve to protect the producing mollusc from the damaging effects of UV radiation. Firstly, the proposed polyene precursors were synthesised using a convergent strategy dependent upon a late-stage Suzuki coupling. Their photochemical, biomimetic conversion into tridachiahydropyrone, phototridachiahydropyrone and oxytridachiahydropyrone, was then accomplished. The interactions of tridachiahydropyrone and its biomimetic precursors with model membrane systems were next explored, using a fluorescence spectroscopic technique. This work demonstrated that the molecules bind to phospholipid vesicles (PLVs) of varying compositions. The synthesis of tridachiahydropyrone within the PLVs was also achieved The propensity of the compounds to act as sunscreens was lastly investigated, by measuring the degree of protection against photochemically-induced lipid peroxidation they conferred on irradiated PLVs, using the thiobarbituric acid reactive substances assay. At high compound concentrations the compounds were found to act as sunscreens, whilst at lower concentrations pro-oxidant activity was observed. In addition to this main work, methodology for the palladium-catalysed cyanation of vinyl halides with acetone cyanohydrin was developed. Conditions were optimised using beta-bromostyrene, and shown to be applicable to a range of diverse substrates. The protocol proved chemoselective for vinyl bromides in the presence of aryl bromides, which were left unaffected and available for further chemical transformations, adding to the synthetic utility of the reaction.
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Shahinian, Sarkis Serge. "Biophysical and functional properties of lipid-modified proteins and model lipopeptides." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40439.
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Three related studies were carried out to understand better the physical properties, cellular function, and means to prepare artificially proteins 'anchored' to membranes by two hydrophobic chains.The first study entailed the development of efficient methodologies to conjugate antibody Fab$ sp prime$ fragments to suitable lipid 'anchors' in liposomes, to allow for the targeted delivery of liposome-associated materials to cells expressing the appropriate cell surface determinant. To monitor the production of Fab$ sp prime$ fragments bearing reactive thiol groups, we developed a maleimide-polyethylene glycol (PEG) conjugate 'thiol reagent' for use in an SDS-PAGE mobility shift assay. Optimizing conditions for Fab$ sp prime$ preparation developed by this approach, we achieved efficient and reproducible coupling of Fab$ sp prime$ fragments to liposomes via a novel lipid 'anchor'. Such Fab$ sp prime$-'targeted' liposomes efficiently delivered encapsulated materials to cultured cells in vitro.
The second study used lipid-modified peptides to investigate the strength of membrane anchoring conferred by dual lipid modifications of the types attached to various intracellular proteins. Based on kinetic measurements of interbilayer transfer of such peptides and peptide-macromolecule conjugates, we suggest that doubly-modified proteins exhibit extremely slow rates of spontaneous intermembrane transfer. We have proposed mechanisms by which this property of 'dual-anchored' proteins may be exploited to achieve efficient subcellular targeting by 'trapping' mechanisms.
The third study sought to assess current models of the mechanism of preferential localization of glycosylphosphatidylinositol (GPI)-proteins to specialized plasma membrane invaginations known as caveolae. This was accomplished using cell membrane-incorporated lipid-PEG-biotin-streptavidin conjugates, whose lateral distribution under various conditions ($ pm$ crosslinking) shows significant overlap with that of endogenous GPI-proteins. We propose a novel mechanistic model based on 'surface-crowding' effects, for the preferential association of lipid-anchored molecules with caveolae, particularly upon crosslinking.
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Wadhwa, Vibhuti. "Biophysical approaches towards greater understanding of eukaryotic zinc sensing." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1593621283583113.
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Ibrahim, Musadiq. "Probing function of unknown proteins by using pharmacophore searching and biophysical techniques." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/3924/.
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The number of protein structures deposited in the Protein data bank is increasing almost exponentially and among these structures many of the proteins are novel with unknown function. Like Docking, Pharmacophore searching is an In-silico technique which is widely used for drug discovery. In pharmacophore searching the main focus is on the hydrogen bond interactions between the ligand and the target protein. The pharmacophore models are generated either by using the already known actives as templates or by utilizing the significant chemical features of the active site. In this thesis the pharmacophore searching has been used to find potential ligands/substrates for unknown proteins and then ligand binding is confirmed by using different biophysical techniques. In the initial phases the pharmacophore models were generated by using Cerius2 and Weblab Viewer pro programs. While in later stages more sophisticated searches were carried out by using DSV (Discovery studio visualizer, Accelrys®). Procedures were optimized for model building by using DSV, which enabled the pharmacophore searching via both the Vector and the Query atom methods. To validate the technique, it was first used on known enzymes with established function e.g. xylose reductase and shikimate kinase. The optimized pharmacophore model when search through the database successfully identified the true substrates for these enzymes among other ligands thereby demonstrating the attainment. The pharmacophore searching technique has been used to find potential ligands for proteins with unknown function on three test cases e.g. TdcF, HutD and PARI. Of the potential pharmacophore hits obtained through database search, a number of compounds were either purchased or synthesised to be tested for binding affinity. Different biophysical techniques like DSC, ITC, CD and NMR were used for this purpose. Among these techniques NMR proved to be the most sensitive technique to differentiate binders from non-binders and to further detect weak and strong bonding in terms of Kd values. For TdcF among other binders the best binder was 2-ketobutyrate with a Kd value of 200µM. In case of HutD, formyl glutamate (Kd = 92µM) and formimino glutamate (Kd = 500µM) came out to be the best binders and could be the true ligands of the protein at physiological concentration. For PARI L-glutamate appeared to be a potential ligand for the protein as confirmed through the NMR experiments. Pharmacophore modelling has been successful in identifying potential interactions provided by the protein active site which in turns specifies the required features to be present in a ligand and later on the successful binding studies further confirm its applicability. In addition, protein structures from the protein data bank (PDB) with unknown ligands (UNK) were identified and manually screened to find examples that could be used to test the applicability of pharmacophore searching methods. The diversity of structures showed that the definition of an unknown ligand is completely inconsistent with many examples where any non spherical density was labelled as unknown ligand and in most cases a single atom is labelled as an unknown ligand, which most likely can be an ion or a water molecule. It appeared that some compounds like glycerol, phosphate and citrate which co-crystallized with the protein due to their presence in the crystallization conditions were also mistakenly assigned as UNK. The pharmacophore method worked successfully in finding suitable ligand (s) for the protein.
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Li, Shi-Jiang. "Biophysical investigations into the secondary structure of Triticum aestivum 5s ribosomal RNA /." The Ohio State University, 1985. http://rave.ohiolink.edu/etdc/view?acc_num=osu148726191911312.
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Stephan, Jules Rabie. "A biophysical and biochemical approach to understanding the interplay between Quaternary structure and function of human calprotectin." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/118277.
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Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2018.Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
In response to an invading pathogen, the host organism initiates an immune response to fight infection. One component of the response involves metal-sequestering proteins that starve pathogens of essential metal nutrients. Humans release calprotectin (CP), a heterooligomer of S100A8 and S100A9, from neutrophils and epithelial cells to prevent microbes from accessing manganese, iron, nickel, and zinc. CP also binds Ca(II) ions, which increases the transition-metal affinity and antimicrobial activity of CP. In addition, Ca(II) causes the S100A8/S100A9 CP heterodimer to form a S100A82/S100A9₂ tetramer. When this dissertation research began, it was known that CP inhibited bacterial growth by sequestering transition metals, and that CP could transmit a proinflammatory signal; however, little was known about the fate of CP after release. The focus of this work was to better understand how the extracellular space may affect CP on biophysical and biochemical levels. Our approach was to study the molecular-level consequences of Ca(II) binding and tetramerization. We found that the heterotetramer exhibited significant resistance to enzymatic proteolysis compared to the heterodimer. Using NMR spectroscopy, we observed that the dynamics of CP change significantly upon Ca(II) binding small, yet notable, alterations in secondary structure. Finally, we discovered that methionine oxidation of CP inhibited Ca(II)-induced tetramerization, resulting in accelerated proteolysis. Taken together, our studies provided new insights into how CP survives the harsh conditions of the extracellular space, and a mechanism for clearing CP from the extracellular space.
by Jules Rabie Stephan.
Ph. D. in Biological Chemistry
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Asiani, Karishma. "Biochemical and biophysical studies on SilE from the sil silver resistance locus." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/39567/.
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Metal ions such as silver (Ag+), mercury (Hg2+), zinc (Zn2+) and copper (Cu+/Cu2+) have a long history of antimicrobial usage and some, such as Cu+/Cu2+, Ag+ and Zn2+ compounds are still used as antimicrobials. Prior to the introduction of antibiotics, Ag+ was arguably the most important antimicrobial and with the rapid emergence of antibiotic resistance, interest in Ag+ and its compounds as alternative antimicrobials have recently been revived. However, resistance to Ag+-based compounds has been emerging, with initial reports of carriage of silver resistance on a Salmonella enetrica serovar Typhimurium multi-resistance plasmid pMG101 isolated from burns patients in 1975. The proposed model for the mechanism of Ag+ resistance encoded by the sil genes from pMG101 involves export of Ag+ ions via an ATPase (SilP), an RND family effluxer (SilCFBA) and a periplasmic chaperone of Ag+ (SilE). SilE is a periplasmic protein predicted to be intrinsically disordered until it binds Ag+ ions. This hypothesis was tested using structural and biophysical studies which showed that SilE is an intrinsically disordered and unstructured protein in its free apo-form, but folds to a compact, defined structure upon optimal binding of six Ag+ ions in its holo-form. Sequence analyses and site-directed mutagenesis established the importance of histidine and methionine containing motifs for Ag+-binding, and identified a nucleation core that initiates Ag+-mediated folding of SilE. The data show that SilE is a molecular metal sponge absorbing up to a maximum of eight Ag+ ions.
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Wright, Thaiesha Andrea. "Advances in Synthesis and Biophysical Analysis of Protein-Polymer Bioconjugates." Miami University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=miami1593472633914607.
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White, Andrew John. "The biophysical chemistry of tooth surfaces : protein and peptide-based technologies for inhibiting erosive tooth wear." Thesis, University of Bristol, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.550331.
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Dental erosion is an increasing problem in many countries around the world, and research in this field has increased dramatically in recent years. Dental erosion is the dissolution of tooth tissues by acids that are not of bacterial origin; most commonly these originate from the diet. Methods to reduce erosion are of great import; the application of milk-derived proteins such as casein and casein-derived proteins are of current interest as anti-erosion agents and are the subject of the work presented here in this thesis. The efficacy of casein and casein-derived proteins as agents to inhibit dissolution of hydroxyapatite in simple citric acid solutions are investigated in chapters 3 and 4 with two different in vitro models. It was found that these proteins inhibit hydroxyapatite dissolution over a range of erosion timescales, concentrations and exposure times. The effect of an in vitro formed salivary pellicle is also examined and the proteins were shown to retain their efficacy. The efficacy of these proteins to inhibit the earliest stages of erosion (surface softening) and more progressed stages of erosion (bulk tissue loss) are investigated in chapter 5 using atomic force microscopy nanoindentation and non-contact optical profilometry respectively. It was found that again these proteins inhibit both surface softening and bulk tissue loss of bovine enamel. The nature of the protective mechanism due to casein is investigated in chapter 6 using a range of complementary, inter-disciplinary techniques such as atomic force microscopy, x-ray reflectometry and sodium dodecyl sulphate polyacrylamide gel electrophoresis. The protective effect is ascribed to a thin protein film, of 6.6 nm in thickness, forming on the mineral surface. In conclusion, casein and casein-derived proteins are shown to have anti-erosion properties and potential as oral healthcare products.
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Patel, Kirti Kantilal. "Biophysical studies on novel metallo-systems and their interactions with DNA and small molecules." Thesis, University of Warwick, 2001. http://wrap.warwick.ac.uk/4169/.
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The DNA binding interactions of five novel bis(2,2':6'2"- terpyridine)ruthenium(II) complexes functionalised with an aryl tail group (i) biphenylene (biphen), (ii) P-napthyl (napth), (iii) phenanthrene (phen), (iv) anthracene (anth) and (v) pyrene (pyr) in the 4' position on each terpyridine ligand were investigated with calf thymus DNA (ct-DNA), poly [d(A-T)]2 and poly [d(G-C)]2 DNA using the spectroscopic techniques of absorbance, circular dichroism (CD) and linear dichroism (LD). All five complexes bind non-covalently to DNA. At low complex concentrations, the anth, phen and pyr complexes were found to intercalate their aryl tail groups between DNA bases. The napth complex exhibits both an intercalative and a non-intercalative mode. The biphen complex exhibits groove binding with no significant tail intercalation. At high metal complex concentrations, aggregation of the complexes on the DNA is observed, resonance light scattering indicate aggregates of low nuclearity along the groove. The DNA binding interactions of a novel series of structurally similar eighteen platinum(H) square planar complexes with subtle ligand variations of the formula [Pt(L)Cl(DMSO)] where L denotes an acylthiourea ligand system were investigated with ct-DNA, poly [d(A-T)]2 and poly [d(G-C)]2 DNA, 5'-mononucleotides and 9- methylguanine. Absorbance, CD and LD studies indicate ligand variation affects DNA binding interaction. Mass spectrometric studies suggest these complexes bind covalently to DNA via the loss of the chloride or possibly the DMSO groups on the platinum. Supramolecular copper(l) and silver(l) metal lo-cyclophanes and their interactions with the isomers of di- and tri-methoxybenzenes and chiral anionic compounds were investigated. The metallo-cyclophanes can adopt two conformations, a helical and a bridging non-helical structure. CD, NMR and X-ray crystallography show the dianion sodium antimonyl-L-tartrate can resolve this mixture. Absorbance and fluorescence studies suggest the metallo-cyclophanes bind methoxybenzenes. The design and synthesis of novel longer asymmetric ligands capable of assembly into a larger suprarnolecular metallo-cyclophanesis included.
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Storti, Barbara. "Interlaced biophysical methods to unveil membrane receptor organization." Doctoral thesis, Scuola Normale Superiore, 2016. http://hdl.handle.net/11384/86202.
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From the introduction: "In my thesis I set out to address these issues by a toolbox of fluorescence imaging techniques such as Förster Resonance Energy Transfer, Fluorescence Anisotropy Imaging, and Spatio-temporal image correlation spectroscopy. I investigated receptor properties such as membrane mobility, microtubule and caveolin-1 binding, and TRPV1 oligomerization status. My experimental strategies benefited from the use of genetically-encodable fluorescent reporters belonging to the green fluorescent protein family."
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Sukthankar, Pinakin Ramchandra. "Biophysical characterization of branched amphiphilic peptide capsules and their potential applications in radiotherapy." Diss., Kansas State University, 2014. http://hdl.handle.net/2097/18174.
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Doctor of PhilosophyDepartment of Biochemistry and Molecular Biophysics
John M. Tomich
Branched Amphiphilic Peptide Capsules (BAPCs) are peptide nano-spheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble in water to form bilayer delimited poly-cationic capsules capable of trapping solutes. We examined the lipid-like properties of this system including assembly, fusion, solute encapsulation, and resizing by membrane extrusion as well as their capability to be maintained at a specific size by storage at 4˚C. These studies along with earlier work from the lab (Gudlur et al. (2012) PLOS ONE 7(9): e45374) demonstrated that the capsules, while sharing many properties with lipid vesicles, were much more robust. We next investigated the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs. We demonstrated that the BAPCs are readily taken up by epithelial cells in culture, escape or evade the endocytotic pathway, and accumulate in the peri-nuclear region where they persist without any apparent degradation. The stability and persistence of the capsules suggested they might be useful in delivering radionuclides. The BAPCs encapsulated alpha particle emitting radionuclides without any apparent leakage, were taken up by cells and were retained for extended periods of time. Their potential in this clinical application is being currently pursued. Lastly we studied the temperature dependence of capsule formation by examining the biophysical characteristics of temperature induced conformational changes in BAPCs and examined the structural parameters within the sequences that contribute to their remarkable stability. A region in the nine-residue sequence was identified as the critical element in this process. The ability to prepare stable uniform nano-scale capsules of desired sizes makes BAPCs potentially attractive as delivery vehicles for various solutes/drugs.
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Hammerstein, Anne Friederike. "Single-molecule chemistry studies with engineered alpha-hemolysin pores." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:1dd1f11d-2b20-42e9-9dfc-c30498822b77.
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Engineered protein nanopores can be used to investigate a wide range of dynamic processes in real time and at the single-molecule level, for example covalent bond making and breaking or the interaction of ligands with their cognate binding sites. The detection of such processes is accomplished by monitoring the current carried by ions through the pore in an applied potential, which is modulated as molecules of interest interact with engineered binding sites within the pore. In contrast to ensemble measurements, where the behaviour of individual molecules is obscured by averaging, single-channel recordings can identify short-lived intermediates and rare reaction pathways, thereby adding to our understanding of fundamental processes in chemistry and biology. The goal of my thesis work was to engineer alpha-hemolysin (αHL) pores to gain insight into such processes. Chapter 1 provides an overview of common techniques used to study single- molecule processes, in particular single channel recordings. General techniques to engineer ion channels and pores are presented, followed by examples of how the alpha-HL pore has been engineered to monitor dynamic processes at the single- molecule level. Chapter 2 describes how alpha-HL pores can be chemically modifeed with a tridentate "half-chelator" ligand. Single channel recordings show that this modifeed pore can be used to determine rates of chelation and the stability of divalent metal ion complexes. The modifeed pore can also be used as a stochastic sensor for the detection of different divalent metal ions in solution. Chapter 3 investigates the chelate-cooperativity between two half-chelator ligands installed in close proximity in the alpha-HL pore, as they form a full complex with a single Zn2+ ion. The single channel recordings reveal a two step process, in which the Zn2+ ion must fiferst bind to one of the two half-chelators, before the second one completes the complex. The rate constants for all the major steps of the process are determined and the extent of cooperativity between the half-chelators is quantifeed. Chapter 4 demonstrates that genetically encoded subunit dimers of alpha-HL can be used to control the subunit arrangement in the heptameric pore. Although techniques exist to prepare heteroheptameric pores, pores containing more than one type of modifeed subunit are not commonly used because it is impossible to distinguish between the permutations of the pore. By using subunit dimers, heptamers in which two defefined subunits are adjacent to each other can be formed, which increases the range of structures that can be obtained from engineered protein nanopores. Chapter 5 explores the possibility of following the nuclease activity of a metal complex in the alpha-HL pore at the single-molecule level. The Rh(III) complex [Rh(bpy)2phzi]2+ binds strongly to CC mismatches in dsDNA, and on activation with UV light promotes the cleavage of one of the two strands. To follow this reaction by single channel recording, a piece of dsDNA with the bound Rh-complex was immobilised in the HL pore and the single current changes under UV irradiation were monitored. The preliminary data indicate that the rate of the photocleavage reaction can be measured.
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Rana, Sandeep. "Synthesis, biophysical analysis and biological evaluation of tricyclic pyrones and pyridinones as anti-alzheimer agents." Diss., Kansas State University, 2009. http://hdl.handle.net/2097/1732.
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Doctor of PhilosophyDepartment of Chemistry
Duy H. Hua
The objectives of this research project were to (i) synthesize different bicyclic and tricyclic pyrone and pyridinone compounds; (ii) study the mechanism of action of these compounds in solution as anti-Aβ (amyloid β) agents using different biophysical techniques; and (iii) study the biological activity of pyrone compounds for the counteraction of Aβ toxicity using MC65 cells, a human neuroblastoma cell line and 5X- familial Alzheimer’s disease (5X FAD, a transgenic mice with five different mutations) mice. A series of tricyclic pyrone and pyridinone compounds were investigated. The tricyclic pyrones and pyridinones were synthesized utilizing a condensation reaction between cyclohexenecarboxaldehye (25) and 4-hydroxy-6-methyl-2-pyone (24) or 4-hydroxy-6-methyl-2-pyridinone (51), respectively. A tricylic pyrone molecule CP2 (2, code name) was synthesized and has an adenine base unit attached to the pyrone core. For structure activity relationship (SAR) studies, the adenine group of CP2 was replaced with other DNA base units (thymine, cytosine and guanine) and various heterocyclic moieties. Since nitrogen containing compounds often exhibit increased bioactivity and brain-penetrating abilities, oxygen atom (O5’) was displaced with a nitrogen atom in the middle ring of the tricyclic pyrone. A condensation reaction of pyrone 51 and 25 was carried out to give the linear pyranoquinoline (52) and the L-shaped pyranoisoquinoline (53). The neurotoxicity of amyloid-β protein (Aβ) is widely regarded as one of the fundamental causes of neurodegeneration in Alzheimer’s disease (AD). Recent studies suggest that soluble Aβ oligomers rather then protofibrils and fibrils may be the primary toxic species. Different biophysical techniques including atomic force microscopy (AFM), circular dichroism (CD), surface plasmon resonance (SPR) spectroscopy, and protein quantification assays were used to study the mechanism of aggregation of Alzheimer Aβ peptide in solution. In search of potentially bioactive compounds for AD therapies, MC65 cell line was used as a screening model. Different tricyclic pyrone and pyridinone compounds protect MC65 cells from death. We studied the efficacy of CP2 in vivo by treatment of 5X FAD mice, a robust Aβ42-producing animal model of AD, with a 2-week course of CP2, which resulted in 40% and 50% decreases in non-fibrillar and fibrillar Aβ species respectively.
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Burley, John Russell. "Pharmacological and biophysical characteristics of voltage-dependant calcium channels in rat cerebellar granule neurones." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264895.
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Buterbaugh, Jeffrey Scott. "The acquisition and analysis of time-resolved fluorescence data from complex systems : applications to problems of photochemical, biophysical, and environmental interest /." The Ohio State University, 1998. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487948807588985.
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Tolbert, Michele M. "Biophysical Characterization of Protein-RNA Interactions Regulating Cap-Independent Translation in Enterovirus 71." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case149874297417556.
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Jeyaharan, Dhadchayini. "Biophysical study into the structure and substrate binding properties of peptido-mimetic ligands to carboxypeptidase G2." Thesis, University of Warwick, 2015. http://wrap.warwick.ac.uk/78855/.
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The antibody directed enzyme pro-drug therapy (ADEPT) is a new anticancer treatment, where pre-clinical experiments concluded that an intermediate step involving the inhibition of carboxypeptidase G2 (CPG2) from the circulatory system prior to pro-drug administration is crucial to prevent systemic toxicity (Bagshawe et al. 1991). The research described in this thesis sought to better understand the mode of binding of these inhibitors to CPG2 using solution state nuclear magnetic resonance (NMR) spectroscopy. A high-yield expression of active and soluble mature CPG2 (in the absence of the leader peptide) in E. coli suitable for NMR studies and co-crystallisation screening is reported. We have used this method to routinely produce milligrams quantities of 1H/13C/15N isotopically-labelled protein suitable for NMR studies. The second aim of this thesis is interactions; interactions between CPG2 and selected inhibitors provided by our industrial partner Mologic Ltd. (Bedford, UK). Different structural parts of the inhibitors were identified by NMR to directly interact with CPG2: the naphthalene and the glutamate groups. Chemical shift perturbations studies show different patterns for CP06 and CP67 inhibitors suggesting that they have different binding mechanisms. Site-directed mutagenesis of residues in P1 pocket of CPG2 reveal no activity against methotrexate (MTX), suggesting that they are key players in substrate recognition, while H285A and E200A mutant proteins display similar activity to wild type CPG2 protein. Although the NMR data described here for CPG2 were incomplete and thus did not yield resonance assignment, we show attempts at a "divide-and-conquer" approach. The CPG2CAT construct shows great promise for downstream NMR studies as it has favourable solution properties and retains key properties of the parent protein, namely enzymatic activity and the ability to self associate.
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Palian, Michael. "Glycopeptide enkephalin analogs: Design, synthesis, biophysical and pharmacological evaluation of potent analgesics with reduced side-effects." Diss., The University of Arizona, 2002. http://hdl.handle.net/10150/280097.
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Pain relief is one of the most fundamental, yet elusive goals of medicine. In the mammalian brain, one means of pain suppression is through activation of the opioid receptors. The opioids themselves are a broad class of centrally acting ligands that may target the delta, kappa, and/or mu opioid receptors in the brain or the spinal column in order to produce pain relief. Glycopeptides have been shown to be versatile, non-toxic alternatives to morphine-induced analgesia in mice. In order to further explore the limits of glycopeptide analgesics, we have synthesized three series of glycopeptide enkephalin analogues (linear glycopeptides, lipo-glycopeptides, and amphipathic alpha-helical glycopeptides) in order to explore the pharmacology and glycopeptide-membrane interactions associated with high-affinity receptor agonism. The three classes of compounds were designed to interact with the cellular membrane to a different degree, and in varying modes of action. Each class of compounds has presented different challenges, but display diverse in vitro and in vivo profiles. Overall, the glycopeptides have shown excellent potential as advanced drug candidates. Total synthesis, circular dichroism, 2-D NMR, molecular modeling, membrane interaction, in vitro binding, in vivo analgesia and dependence liability studies have been performed and will be discussed.
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Miller, Effie Kisgeropoulos. "From Structure to Function: Utilizing the Biophysical Toolbox to Interrogate a Novel Class of Mn/Fe Proteins." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1588957858885161.
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Lindström, Fredrick. "Biological membrane interfaces involved in diseases : a biophysical study." Doctoral thesis, Umeå universitet, Kemi, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-806.
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Interactions between peptides and biological lipid membranes play a crucial role in many cellular processes such as in the mechanism behind Alzheimer’s disease where amyloid-beta peptide (Abeta)is thought to be a key component. The initial step of binding between a surface active peptide and its target membrane or membrane receptor can involve a non specific electrostatic association where positively charged amino acid residues and a negatively charged membrane surface interact. Here, the use of high resolution MAS NMR provides a highly sensitive and non perturbing way of studying the electrostatic potential present at lipid membrane surfaces and the changes resulting from the association of peptides. The interaction between pharmacologically relevant peptides and lipid membranes can also involve incorporation of the peptide into the membrane core and by complementing the NMR approach with differential scanning calorimetry (DSC) the hydrophobic incorporation can be studied in a non invasive way. By using 14N MAS NMR on biological lipid systems for the first time, in addition to 31P, 2H NMR and differential scanning calorimetry (DSC), gives a full picture of the changes all along the phospholipid following interactions at the membrane interface region. Being able to monitor the full length of the phospholipid enables us to differentiate between interactions related to either membrane surface association or hydrophobic core incorporation. This approach was used to establish that the interaction between nociceptin and negatively charged lipid membranes is electrostatic and hence that nociceptin can initially associate with a membrane surface before binding to its receptor. Also, it was found that Abeta can interact with phospholipid membranes via two types of interactions with fundamentally adverse effects. The results reveal that Abeta can associate with the surface of a neuronal membrane promoting accelerated aggregation of the peptide leading to neuronal apoptotic cell death. Furthermore it is also shown that Abeta can anchor itself into the membrane and suppress the neurotoxic aggregation of Abeta.
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Yang, Dazhou. "Synthesis and biophysical evaluation of thiazole orange derivatives as DNA binding ligands." Scholarly Commons, 2014. https://scholarlycommons.pacific.edu/uop_etds/141.
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Guanine-rich telomeric DNA at the end of chromosomes can form a unique DNA tertiary structure - G-quadruplex, which is known to inhibit the binding of telomerase to telomeric regions in cancer cells and thus regulate unrestricted cancer cell growth. Hence, G-quadruplex DNA has recently become a promising target in oncology. The formation of G-quadruplex structures is greatly facilitated by G-quadruplex binding ligands such as Thiazole orange (TO). Compared with other G-quadruplex binding ligands, TO has an intriguing tunable fluorescence property. Upon binding to DNA, the fluorescence of TO can increase up to 1000-fold, making it an attractive probe for studying ligand-DNA interactions. However, the poor binding affinity and minimal binding selectivity towards different DNA conformations greatly limit its applications. My research focuses on developing G-quadruplex binding ligands using TO as a scaffold. In the first part of this work, we investigated the feasibility of increasing the TO binding affinity and selectivity toward G-quadruplex DNA by introducing side chains to the molecule. TO derivatives containing various side chains were successfully synthesized and characterized. Biophysical and biochemical studies with duplex and G-quadruplex DNA showed that tethering side chains to TO is an effective approach to tune its ability of binding to duplex or G-quadruplex DNA. Possible binding modes of the effective derivatives were studied using AutoDock. Their inhibition of telomerase activities was studied using the TRAP assay. The cytotoxicity of these derivatives toward three cancer cell lines was also investigated using the MTS assay. The second part of this work focuses on development of TO-based G-quadruplex DNA binding ligands that can bind to DNA via the dual recognition mode. TO was tethered with pyrene, naphthalene diimide, and anthraquinone respectively to yield three novel conjugates. Further investigation suggested that the conjugate of TO with naphthalene diimide (TO-NF) gave the best G-quadruplex binding affinity. It binds to G-quadruplex DNA via the end stack mode and strongly inhibits the telomerase activity. The cytotoxicity results will also be discussed in this presentation.
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Wang, Yin 1951. "Influences of membrane biophysical properties on the Metarhodopsin I to Metarhodopsin II transition in visual excitation." Diss., The University of Arizona, 1997. http://hdl.handle.net/10150/282520.
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Current biophysical studies of membrane proteins are centered on the relation of their structures to key biological functions of membranes in terms of lipid-protein interactions. The conformational transition of rhodopsin from Metarhodopsin I to Metarhodopsin II (Meta I-Meta II) is the triggering event for the visual process. Meta II is the activated form of the visual receptor and binds a signal transducing G protein (transducin), followed by two amplification stages which lead to generation of a visual nerve impulse. Herein, flash photolysis and surface plasmon resonance (SPR) spectroscopy techniques have been used to monitor the Meta I-Meta II transition of rhodopsin in various membrane recombinants. The flash photolysis experiments clearly show a substantial shift to the left of the Meta I-Meta II equilibrium for rhodopsin in egg phosphatidylcholine recombinant membranes. Investigation of the influences on rhodopsin function by non-lamellar forming lipids reveals a characteristic relationship between the Gibbs free energy change for the Meta I-Meta II equilibrium of rhodopsin and the intrinsic curvature of the lipid bilayer. Complementary SPR studies suggest a protrusion of the protein at the activated Meta II state which may be associated with exposure of recognition sites for the signal transducing G protein on the cytoplasmic surface of rhodopsin. All the experimental results obtained here are consistent with the hypothesis of a new flexible surface biomembrane model. The Meta II state is favored by a negative spontaneous curvature of the bilayer, corresponding to an imbalance of the lateral forces within the polar head groups and acyl chains. The mean curvature of membrane bilayer in the Meta II state reflects the small spontaneous curvature of the lipid bilayer in the vicinity of protein. Relief of the lipid curvature frustration in the Meta II state energetically couples the lipids to the photoexcitation of rhodopsin. Consideration of the various energetic contributions suggests the bilayer curvature free energy provides a reservoir of work in the modulation of rhodopsin function in the visual process. These studies that biophysical properties of the liquid-crystalline lipid bilayer are important in relation to protein function and may be relevant to the biomedical investigations of visual dysfunction.
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Gaspar, Diana Maria Diez. "A biophysical approach to Phospholipase A2 activity and inhibition by non-steroidal anti-inflammatory drugs." Doctoral thesis, Faculdade de Farmácia da Universidade do Porto, 2009. http://hdl.handle.net/10216/63806.
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