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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
71

Cloning and characterization of the receptor for interferon-tau

Han, Chun-Sheng, January 2000 (has links)
Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Title on signature page has the Greek symbol for tau. Typescript. Vita. Includes bibliographical references (leaves 167-196). Also available on the Internet.
72

Structure and mode of action of the TolA-TolB complex from Pseudomonas aeruginosa

Holmes, Peter January 2016 (has links)
Protein-protein interactions (PPIs) across the cell envelope of Gram-negative bacteria are critical for mediating signal transduction pathways that underpin cellular homeostasis. The Ton and Tol Pal systems are two conserved, ancestrally related protein networks that are also required for bacterial pathogenesis. Both Ton and Tol-Pal traverse the periplasm to effect different functions at the outer membrane (OM). Tol-Pal is composed of a homologous complex of three inner membrane proteins, TolQ-TolR-TolA (linked to proton motive force) and two additional periplasmic proteins TolB and Pal. The physiological role of the Tol-Pal system is to stabilise the OM, however the mechanism involved is unknown. TolA is however known to form a crucial protein-protein interaction via its C-terminus with the disordered N-terminus of TolB. Prior to this thesis, determination of the molecular features underlying a protein-protein complex between TolA and an endogenous binding partner TolB had never been accomplished. In this work, I describe the first structure comprising the TolA-TolB complex from Gram negative bacteria. The structure of this complex was determined from Pseudomonas aeruginosa by solution NMR spectroscopy. I determined the interaction between P. aeruginosa TolA and a TolB N terminal peptide to be relatively weak using fluorescence anisotropy. I found that TolB interacts with TolA through an analogous mechanism to that seen in TonB-dependent transporters. Based on these studies and bioinformatics analyses, I hypothesize that the evolutionary resilience of the Tol-Pal system to external pressures is contingent on the preservation of the TolA-TolB interface. Structure-based mutations within the TolA-TolB complex were also evaluated for their effect on in vivo function of the Tol-Pal complex and impact on complex formation in vitro. Taken together, the results demonstrate that protein networks which transduce energy to the OM through PMF-dependent systems in bacterial cells appear to follow a common β-strand augmentation mechanism.
73

Identification and analysis of ligand binding sites by computational mapping

Ngan, Chi Ho January 2012 (has links)
Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Ligand binding sites in proteins generally include "hot spots" that contribute a large fraction of the binding free energy and, therefore, are of prime interest in drug design. To find hot spots on the protein surface, a protein can be screened against libraries of small organic molecules to identify interaction sites using nuclear magnetic resonance (NMR) spectroscopy or the X-ray crystallographic technique Multiple Solvent Crystal Structures (MSCS). Small organic molecules can bind at several locations on the surface of a protein, but many different molecules congregate only in "consensus sites" identifying the hot spots. The mapping algorithm FTMAP is a computational analogue of experimental fragment screening methods. The principles of computational mapping were used for the development and testing of the binding site identification algorithm FTSITE, implemented as a web-based server. Finding ligand binding sites in silico is a classical challenge, and the success rate of identifying the ligand binding site as the first predicted site has increased to 83% during the last decade. FfSITE, based on biophysical modeling of protein-ligand interactions, increased the success rate to 94% on the same established test sets. Critical to the success of FfSITE is the use of multiple small molecules as probes; screening by X-ray crystallography and NMR spectroscopy had demonstrated a tendency of ligand binding sites to bind small organic compounds ranging 1n shapes, sizes, and polarities. Further, FfSITE does not use surrogate measures of ligand binding propensity such as site geometries and dimensions. It was shown that FTSITE can also successfully identify allosteric ligand binding sites that can serve as candidates for drug design. Furthermore, the hot spot information provided by FfMAP was shown to guide the development of core fragments, found by experimental fragment screening , into optimal ligands for a number of drug target proteins. Computational mapping can also be used for fragment-based drug design by finding fragments with preference for some regions of the binding site. To facilitate this analysis , a server enabling the fast generation of force field parameters for user-specified small molecules or fragments was developed. / 2031-01-02
74

Continuous-flow dynamic dialysis and its application to collagen-ligand interactions

Sparrow, Neil Arthur January 1983 (has links)
Studies undertaken to investigate the binding of low molecular mass analogues of polyphenolic vegetable tannins to collagen have prompted the development of a new method to investigate protein-ligand interactions. This method, the continuous-flow dynamic dialysis method (CFDD), differs from conventional dialysis procedures used for protein-ligand binding studies. In this method, the ligand concentration in the diffusate is monitored automatically at successive closely spaced time intervals while being continuously eluted from the dialysis cell. The primary data obtained by this method consists of a series of spectrophotometric absorbance measurements representing the ligand concentration in the sink compartment of a dialysis cell. This primary data is recorded by means of a data logging device onto a punched paper tape for subsequent computer processing. Two original methods are presented for analysing the primary data to extract the protein-ligand binding isotherm. The first of these is a direct analysis which relies on Fick's first law of diffusion. In this method it is necessary to establish, by means of a control experiment, a value for the ligand permeation constant. This is used in a subsequent analysis to establish a relationship between the measured rate of diffusion of the ligand from a protein-ligand mixture and the concentration of unbound ligand which is in equilibrium with the protein-ligand complex. The protein-ligand binding isotherm is obtained from parametric equations which give the quantity of ligand bound to the protein and the concentration of unbound ligand in the sample compartment as functions of time. The second method, which is more general, utilizes the same primary data but is based on establishing a system transfer function to characterise the dialysis and eluting processes. This analysis depends on the linearity of the system and utilizes numerical laplace transforms of the primary data sets obtained from control and protein-ligand dialyses. Laplace transforms are used to effect a deconvolution of the transfer function from the primary data and yield the concentration of ligand in equilibrium with the protein-ligand complex. This procedure yields, simultaneously, both the total ligand concentration and the concentration of unbound ligand in the protein compartment of the dialysis cell. These quantities are used to establish the binding isotherm for the protein ligand system. Numerical inversion of the laplace transforms in this analysis is effected by their reduction to Fourier series. The experimental reliability of the continuous-flow dynamic dialysis method, and validity of the two analytical methods used to derive a binding isotherm from dialysis data are evaluated from studies of the binding of phenol red to bovine serum albumin (BSA) at 15⁰, 20⁰ and 25⁰ C, as well as from simulated binding curves generated by the numerical solution of the differential equations used to describe the dialysis and elution process in terms of a two-site Scatchard binding model. The method is used to investigate the binding to collagen of a series of low molecular mass phenolic compounds which can be isolated from Wattle and Quebracho vegetable tannin extracts. These compounds can be considered as monomeric precursor analogues of the polymeric vegetable tannins. The binding of these ligands to collagen is shown to be characterised by high capacity, low affinity binding in which the uptake of ligand by the protein increases linearly with increasing ligand concentration. Collagen exhibits no indication of site saturation for these ligands over the experimentally accessible concentration ranges investigated.
75

Diphosphine Ligand Substitution in H4Ru4(CO)12: X-ray Diffraction Structures and Reactivity Studies of the Diphosphine Substituted Cluster Products

Kandala, Srikanth 12 1900 (has links)
The tetraruthenium cluster H4Ru4(CO)12 has been studied for its reactivity with the unsaturated diphosphine ligands (Z)-Ph2PCH=CHPPh2, 4,5-bis (diphenylphosphino)-4-cyclopenten-1,3-dione, bis(diphenyphosphino)benzene and 1,8- bis(diphenyl phosphino)naphthalene under thermal, near-UV photolysis, and Me3NO-assisted activation. All three cluster activation methods promote loss of CO and furnish the anticipated substitution products that possess a chelating diphosphine ligand. Clusters 1, 2, 3 and 4 have been characterized in solution by IR and NMR spectroscopies, and these data are discussed with respect to the crystallographically determined structures for all new cluster compounds. The 31P NMR spectral data and the solid-state structures confirm the presence of a chelating diphosphine ligand in all four new clusters. Sealed NMR tubes containing clusters 1, 2, 3 and 4 were found to be exceeding stable towards near-UV light and temperatures up to ca. 100°C. The surprisingly robust behavior of the new clusters is contrasted with the related cluster Ru3(CO)10(bpcd) that undergoes fragmentation to the donor-acceptor compound Ru2(CO)6(bpcd) and the phosphido-bridged compound Ru2(CO)6 (µ-PPh2)[µ-C=C(PPh2)C(O)CH2C(O)] under mild conditions. The electrochemical properties have been investigated in the case of clusters 1 and 2 by cyclic voltammetry, and the findings are discussed with respect to the reported electrochemical data on the parent cluster H4Ru4(CO)12.
76

Kinetic Studies on C‐h Bond Activation in the Reaction of Triosmium Clusters with Diphosphine and Amidine Ligands

Yang, Li 12 1900 (has links)
The reaction of 1-(diphenylphosphino)-2-(diphenylphosphito)benzene (PP*) and Os3(CO)10(ACN) has been investigated. A combined experimental and computational study on the isomerization of 1,2-Os3(CO)10[μ-1,2-Ph2P(C6H4)P(OPh)2] (A) and 1,1-Os3(CO)10[μ-1,2-Ph2P(C6H4)P(OPh)2] (B) and reversible ortho-metalation exhibited by the triosmium cluster B are reported. The subsequent conversion of cluster B to the hydrido cluster HOs3(CO)9[μ-1,2-PhP(C6H4-η1)C6H4P(OPh)2] (E) and the benzyne-substituted cluster HOs3(CO)8(µ3-C6H4)[μ-1,2-PhP(C6H4)P(OPh)2] (N) has been established. All of these new clusters have been isolated and fully characterized in solution by IR and NMR spectroscopy; in addition, X-ray diffraction analyses have been performed on the clusters A, B, J, and N. The ortho-metalation reaction that gives cluster E is shown to be reversible, and the mechanism has been probed using selectively deuterated PP* isotopomers. Kinetic and thermodynamic isotope data, in conjunction with DFT calculations, are presented that support the existence of an intermediate unsaturated cluster in the ortho-metalation reaction. Due to interest in the coordination chemistry of formamidines, the non-symmetric amidine ligands PhNC(Me)NHPri, PhNC(Et)NHPri, and (2,4,6-Me3C6H2)NC(Me)NHPri, have been synthesized, and their reaction with Os3(CO)10(MeCN)2 has been investigated. Of the twelve new clusters prepared in section, seven have been structurally characterized by X-ray crystallography.
77

Protein – Ligand Binding: Estimation of Binding Free Energies

Ranganathan, Anirudh January 2012 (has links)
Accurate prediction of binding free energies of protein-ligand system has long been a focus area for theoretical and computational studies; with important implications in fields like pharmaceuticals, enzyme-redesign, etc. The aim of this project was to develop such a predictive model for calculating binding free energies of protein-ligand systems based on the LIE-SASA methods. Many models have been successfully fit to experimental data, but a general predictive model, not reliant on experimental values, would make LIE-SASA a more powerful and widely applicable method. The model was developed such that There is no significant increase in computational time No increase in complexity of system setup No increase in the number of empirical parameters. The method was tested on a small number of protein-ligand systems, selected with certain constraints. This was our training set, from which we obtain the complete expression for binding free energy. Expectedly, there was good agreement with experimental values for the training set On applying our model to a similar sized validation set, with the same selection constraints as for the training set, we achieved even better agreement with experimental results, with lower standard errors. Finally, the model was tested by applying it to a set of systems without such selection constraints, and again found good agreement with experimental values. In terms of accuracy, the model was comparable to a system specific empirical fit that was performed on this set. These encouraging results could be an indicator of generality.
78

Engineering a better receptor: characterization of retinoid x receptor alpha and functional variants

Watt, Terry J. 14 November 2007 (has links)
The human retinoid X receptor alpha (hRXRalpha) is a member of the nuclear receptor super-family of ligand-activated transcription factors. The Doyle laboratory has previously engineered a variety of functional hRXRalpha variants that activate gene expression in response to synthetic ligands (LG335 and γ-oxo-1-pyrenebutyric acid), compounds that are poor activators of wild-type hRXRalpha. The variants generally no longer respond to the wild-type ligand 9-cis retinoic acid. To enable targeting of these engineered receptors to arbitrary DNA sequences, we developed a program, ESPSearch, for identifying short or specific sequences in DNA or protein. ESPSearch enables identification of combinations of known zinc finger motifs to target arbitrary genes, as well having several other applications. The ability to target any DNA sequence means that the engineered receptors can be directed to control any gene. The ligand binding, self-association, coactivator interactions, and unfolding properties of the ligand binding domain of wild-type hRXRalpha were characterized. Our expression and purification protocol improves upon existing methods, providing high purity protein in a single step with more than twice prior yields. A general fluorescence-based method for measuring ligand affinity with hRXRalpha was developed, and used to determine binding constants for the small molecules. The presence of a peptide containing the binding motif from coactivator proteins (LxxLL) differentially increased the affinity of the receptor for the ligands. Assays to determine the self-association give a Kd for the dimer-tetramer equilibrium of 35 µM. hRXRalpha was found to denature irreversibly when heated, but shifts in apparent Tm due to ligands correlates strongly with the ligand binding affinities. Our results clarify disparities in existing reports and provide a benchmark for comparison. Reliable analysis of our data led to the development of a computer program for rigorous, automated data fitting. Nine functional variants of hRXRalpha were characterized to probe correlations between biophysical properties and the observed functional activity of the receptors, which differ significantly from wild-type. Although the correlation between ligand binding affinity and melting temperature was strong for all variants, there was essentially no correlation between ligand binding and activation of the variants. The mutations, which are all contained within the binding pocket, have significant long-range effects on the protein, causing changes in ligand-LxxLL interactions and oligomerization of the variants. Experimental and computational analysis of selected mutations suggests that they are highly coupled, complicating protein design. However, the large variation in properties amongst the variants also suggests that hRXRalpha can be mutated extensively while still retaining function. The long-range impact of binding pocket mutations will need to be taken into account in future engineering projects, as hRXRalpha is a flexible, dynamic protein.
79

Kinetic analysis of Fcγ receptor and T cell receptor interacting with respective ligands

Jiang, Ning 12 August 2005 (has links)
Low affinity Fcg receptor III (FcgRIII, CD16) triggers a variety of cellular events upon binding to the Fc portion of IgG. A real-time flow cytometry method was developed to measure the affinity and kinetics of such low affinity receptor/ligand interactions, which was shown as an easily operated yet powerful tool. Results revealed an unusual temperature dependence of reverse rate of CD16aTM dissociating from IgG. Except for a few studies using mammalian cell CD16s, most kinetics analyses use purified aglycosylated extracellular portion of the molecules, making it impossible to assess the importance of the receptor anchor and glycosylation on ligand binding. We used a micropipette adhesion frequency assay to demonstrate that the anchor length affects the forward rate and affinity of CD16s for IgG in a species specific manner, most likely through conformational changes. Receptor glycosylation dramatically reduced ligand binding by 100 folds. T cell receptor (TCR) is arguably the most important receptor in the adaptive human immune system. Together with coreceptor CD4 or CD8, TCR can discriminate different antigen peptides complexed with major histocompatibility complex (MHC) molecule (pMHC), which differ by as few as only one amino acid, and trigger different T cell responses. When T cell signaling was suppressed, TCR had similar affinity and kinetics for agonist and antagonist pMHC whose binding to CD8 was undetectable. TCR on activated T cell had a higher affinity for pMHCs, suggesting that TCRs organize themselves differently on activated T cells than on naïve T cells. In the absence of inhibitors for signaling, TCR binds agonist pMHC with several orders of magnitude higher affinity than antagonist pMHC. In addition, engagement of TCR by pMHC signals an upregulation of CD8 binding to pMHC, which is much stronger than the TCR-pMHC binding. The transition from weak TCR binding to the strong CD8 binding takes place around 0.75 second after TCR in contact with pMHC and can be reduced by several inhibitors of tyrosine and lipid phosphorylation, membrane rafts, and actin cytoskeleton. These results provide new insights to understanding T cell discrimination.
80

Receptor concentration affects glucocorticoid action

Robertson, Steven Ernest 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2011. / See also the post-print version of the article that was published from the PhD - http://hdl.handle.net/10019.1/19557 / ENGLISH ABSTRACT: Glucocorticoid receptor (GR) levels, which modulate the response to glucocorticoids (GCs), vary between tissues and individuals and are altered by physiological and pharmacological effectors. In this study we set out to investigate the effects and implications of differences in GR concentration. Firstly, we established conditions that resulted in three statistically different GR populations in transiently transfected COS-1 cells. We demonstrated, using whole cell saturation ligand binding experiments, that high levels of wild type GR, but not of dimerization deficient GR, exhibited positive cooperative ligand binding with a concomitant increased ligand binding affinity. Furthermore, we established, through co-immunoprecipitation and fluorescent resonance energy transfer, that ligand independent dimerization correlates with positive cooperative ligand binding. This is the first time that positive cooperative ligand binding and increased ligand binding affinity have been explicitly correlated and linked to increased ligand independent dimerization of the GR. The downstream consequences of variation in GR concentration and dimerization included modulation of GR import and export rates, as investigated through live cell as well as immunofluorescent analysis. Furthermore, the nuclear distribution of GR was also influenced by GR dimerization. The major function of the GR is as a transcription factor, which mediates the response to GCs via activation or repression of genes. We have revealed direct influences of GR concentration and dimerization in a number of promoter reporter assays as well as in the transactivation of an endogenous gene. Specifically, cooperative ligand binding was found to be responsible for the GR level dependent potency shift in transrepression of an NF B containing promoter reporter construct via dexamethasone and the shift in the bio-character of Compound A, a dissociative GR agonist. Transactivation potency of dexamethasone as well as the partial agonist bio-character of medroxyprogesterone and mifepristone via a multiple GRE containing promoter reporter construct were influenced directly by cooperative ligand binding. Dimerization of the GR was shown to be crucial for ligand dependent transactivation of a single GRE containing promoter reporter construct, while ligand independent transactivation of both single and multiple GRE containing constructs was significantly increased due to an increase in GR concentration. The endogenous GC responsive glucocorticoid induced leucine zipper (GILZ) gene demonstrated significant ligand independent transactivation at GR levels, which displayed ligand independent dimerization. An increase in GR concentration resulted in an increase in efficacy through all promoter reporter constructs as well as the endogenous GILZ gene. Positive cooperative binding and the concomitant increase in ligand binding affinity to the GR at high levels may be a crucial factor in determining both the efficacy and potency of the GC response. Considering the significant differences in GR concentrations expressed by different tissues and by individuals within the same tissue, our findings may explain the interindividual as well as tissue specific responses to GC treatment and suggest an important mechanism of action through which the GR is primed to responsed to subsaturating GC concentrations and displays a significant level of ligand independent activity. / AFRIKAANSE OPSOMMING: Glukokortikoïed reseptor (GR) vlakke, wat die gedrag van glukokortikoïede (GCs) moduleer, wissel tussen weefsels en onder individue en word verander deur fisiologiese en farmakologiese effektore. In hierdie studie ondersoek ons die gevolge en implikasies van verskille in GR konsentrasie. Eerstens het ons die kondisies vasgestel wat benodig word om drie statisties verskillende GR populasies te vestig in kortstondige getransfekteerde COS-1 selle. Ons het getoon, met behulp van die heel sel versadigings ligand bindings eksperimente, dat hoë vlakke van wilde-tipe GR, maar nie van dimeriserings gebrekkige GR, positiewe koöperatiewe ligand binding, met 'n gepaardgaande toename in ligand bindings affiniteit, toon. Verder het ons bevestig, deur ko-immunopresipitasie en fluoressente resonansie energie-oordrag, dat ligand onafhanklike dimerisering korreleer met positiewe koöperatiewe ligand binding. Dit is die eerste keer dat positiewe koöperatiewe ligand binding en verhoogde ligand bindings affiniteit uitdruklik gekorreleer en gekoppel word aan verhoogde ligand onafhanklike dimerisering van die GR. Die daarop nagevolge van variasie in GR konsentrasie en dimerisering sluit in modulasie van die invoer en uitvoer tempo van die GR, soos ondersoek deur lewendige sel sowel as immunofluorescente analise. Verder is die verspreiding van die GR in die kern ook beïnvloed deur GR dimerisering. Die belangrikste funksie van die GR is as 'n transkripsie faktor, wat die respons van GCS bemiddel via aktivering of onderdrukking van gene. Ons het die direkte invloed van GR konsentrasie en dimerisering in 'n aantal promotor verslaggewer essais sowel as in die transaktivering van endogene gene onthul. Spesifiek, is gevind dat koöperatiewe ligand binding verantwoordelik is vir die GR vlak afhanklike verskuiwing in transrepressie potensie van 'n NF B bevattende promotor verslaggewer konstruk via deksametasoon en die verskuiwing van die biokarakter van verbinding A,' dissosiatiewe GR agonis. Transaktiverings potensie van deksametasoon, asook die gedeeltelike agonis bio-karakter van medroksie-progesteroon en mifepristoon, via 'n veelvoudige GRE bevattende promotor verslaggewer konstruk is direk beïnvloed deur koöperatiewe ligand binding. Dimerisering van die GR is getoon om deurslaggewend vir ligand afhanklike transaktivering van 'n enkele GRE bevattende promotor verslaggewer konstruk te wees, terwyl ligand onafhanklike transaktivering van beide enkel-en veelvoudige GRE bevattende konstrukte aansienlik toegeneem het as gevolg van toename in GR konsentrasie. Die endogene GC responsiewe glukokortikoïed geïnduseerde leusien rits (GILZ) gene het beduidende ligand onafhanklike transaktivering gedemonstreer op GR vlakke wat ligand onafhanklike dimerisering toon. 'n toename in GR konsentrasie het gelei tot toename in die effektiwiteit van al die promotor verslaggewer konstrukte, sowel as die endogene GILZ gene. Positiewe koöperatiewe ligand binding en die gepaardgaande toename in ligand bindings affiniteit van die GR by hoë vlakke kan 'n belangrike faktor wees in die bepaling van sowel die effektiwiteit as die potensie van die GC respons. As die aansienlike verskille in GR konsentrasies van verskillende weefsels en tussen verskillende individue in dieselfde weefsel in ag geneem word, kan ons bevindings die inter-individuele sowel as weefsel spesifieke response op GC behandeling verduidelik en stel dit 'n belangrike meganisme van aksie voor waardeur die GR voorberei word om op sub-versadigings konsentrasies van GC te reageer deur 'n beduidende vlak van ligand onafhanklike aktiwiteit te toon.

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