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1
Baas, Tracey Lynn. "The design, synthesis, and characterization of template assembled synthetic proteins /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/11561.
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Robertson, Nicola. "Protein synthesis." Thesis, University of Edinburgh, 1996. http://hdl.handle.net/1842/14314.
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Reyes, Samuel Onofre J. "Expanding beta-turn analogs for mimicking protein-protein hot spots." Thesis, [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1748.
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Hassan, Hani Mutlak Abdullah. "Chemical Synthesis of Protein Ligands." Thesis, University of Manchester, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501975.
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Bland, Lorraine. "Methodology for chemical protein synthesis." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/12014.
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A number of groups have been investigated as potential protecting groups for the thiol functionality of cysteine residues during Solid Phase Peptide Synthesis. Of these the 4-picolyl system has been found to be successful. A short, efficient synthesis to the protected amino acid and mild cleavage conditions have been developed. This protected amino acid has been applied in the synthesis of a number of interesting cysteine-containing peptide targets. Deglycosylated human erythropoietin (dhEPO) has been synthesised by SPPS, employing the picolyl group for cysteine protection. Purification of this material has been achieved by gel filtration. The synthetic material has been characterised by amino acid analysis, SDS-PAGE, iso-electric focusing and N-terminal sequencing. Attempts have been made to synthesise deglycosylated human erythropoietin via a fragment coupling technique. All fragments were successfully synthesised and purified and model ligation studies carried out.
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Gordon, Carolyn Alexandra. "Investigations in chemical protein synthesis." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/14919.
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The chemical synthesis of the 184aa anti-angiogenic peptide, endostatin, was undertaken. Preparation via the stepwise of two large fragments approximately 90aa in length was attempted but was unsuccessful. A method that would allow the efficient, sequential coupling of several small fragments was required. To this end, the segment coupling of minimally protected fragments via transfer active ester condensation (TAEC), a technique recently developed within this research group, was investigated. The fragments for synthesis were provisionally selected based on hydrophobicity and potential coupling sites. These peptide fragments were then optimised for stepwise solid phase peptide synthesis (SPPS), using the Fmoc strategy. Peptides containing two types of C-terminal functionality - hydrazides and semicarbazides - were prepared. An alternative strategy for the synthesis of the Wang resin-based hydrazide linker, first proposed by Wang and Merrifield in 1969, was developed to facilitate this process. Peptide fragments of up to 20aa in length were successfully coupled using TAEC. A novel approach to the protection of arginine side chains was also investigated. This target was based on the dibenzocycloheptenyl system, a species which was originally deigned for use as a linker in the preparation of peptide amides. Recent work by Noda involved a derivative of this linker, which has proven to be significantly more acid labile than current arginine protection. Concurrent work on an improved design led to an alternative system - the dimethoxy-suberyl compound - being proposed. The synthesis of the target molecule was achieved in seven steps; key steps involved a Perkin reaction and an intramolecular Friedel Krafts acylation. Coupling to arginine was undertaken but proved unsuccessful.
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Dorywalska, Magdalena. "Conformational dynamics of protein synthesis /." May be available electronically:, 2007. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Badger, David B. "Design and Synthesis of Protein-Protein Interaction Inhibitor Scaffolds." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/3964.
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Many currently relevant diseases such as cancer arise from altered biological pathways that rely on protein-protein interactions. The proteins involved in these interactions contain certain functional domains that are responsible for the protein's biological activities. These domains consist of secondary structural elements such as α-helices and Β-sheets which are at the heart of the protein's biological activity. Therefore, designing drugs that inhibit protein-protein interactions by binding to these key secondary structural elements should provide an effective treatment for many diseases. Presented in this dissertation are the designs, syntheses, and biological evaluations for both novel α-helix and novel Β-sheet mimics.
The α-helix mimics were designed to inhibit the interactions between the tumor suppressor protein p53 and its inhibitor protein, MDM2. We also targeted the interactions between the Bak/Bcl-xL proteins. Using the knowledge gained from Hamilton's 1,4-terphenylene scaffold, we designed our inhibitors to be non-peptidic small molecule α-helix mimics. These molecules were designed to bind to the NH2-terminal domain of MDM2 protein thus preventing it from binding to the p53 protein thereby allowing p53 to induce apoptosis. The α-helix mimetic scaffold is designed around a central functionalized pyridazine ring while maintaining the appropriate distances between the ith, ith+4, and ith+7 positions of a natural alpha helix.
The Β-sheet mimics were designed as inhibitors for the integrin mediated extracellular matrix cell adhesion found in Multiple Myeloma. We have designed, synthesized, and incorporated novel Β-turns to induce the formation of Β-hairpins as well as to cyclize the peptides in order to increase their binding affinities and reduce proteolytic cleavage. Given that many protein-protein interactions occur through hydrophobic interactions; our primary Β-turn promoter was designed with the ability to alter the Β-hairpin's hydrophobicity depending on the sulfonyl group used in the turn. The synthesis of several different sulfonyl chlorides for use in our Β-turn promoter is included in this section. We have also provided a detailed structural analysis and characterization of these new cyclic peptides via NMR and CD spectrometry. Using standard 2D NMR methods, we have elucidated the 3D conformation of several peptides in solution. We have also studied the structure activity relationships (SAR) for these cyclic peptides and then correlated these results with those obtained from the NMR studies.
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Blum, Janna Karen. "Broadening the enyzme-catalyzed synthesis of semi-synthetic antibiotics." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/39528.
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An alpha-amino ester hydrolase (AEH) applicable to synthesis of semi-synthetic antibiotics was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913. AEHs catalyze the synthesis and hydrolysis of alpha-amino beta-lactam antibiotics. The enzyme was characterized for thermodynamic and kinetic parameters. The enzyme shows optimal ampicillin hydrolytic activity at 25C and pH 6.8. The AEH enzymes have been shown to have excellent synthetic capability. Additionally, we demonstrated the first fully aqueous enzymatic one-pot synthesis of ampicillin direct from the natural product penicillin G eliminating the isolation of the intermediate 6-APA. Lastly, to improve the thermostability of the AEH a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-factors from the available crystal structures of the known AEH from Xanthomonas citri. Our best variant, which is a quadruple mutant, E143H/A275P/N186D/V622I, which has a T_50_30, the temperature at which the half-life is 30 minutes, of 34C and 1.3-fold activity compared to wild-type. Overall, we have successfully improved the understanding of the AEH class of enzymes and applied a novel cascade application, demonstrating AEHs unique applicability in the synthesis of beta-lactam antibiotics. The improved thermostability will further improve the industrial relevance of AEHs.
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Ferris, Douglas K. "Protein phosphatases and protein kinases in dictyostelium." Diss., Virginia Polytechnic Institute and State University, 1986. http://hdl.handle.net/10919/50017.
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In the present work, the chromatographic behavior and partial characterization of 5 protein phosphatases, 6 paranitrophenyl phosphate (pnpp) phosphatases and 2 protein kinases from Dictyostelium are described. .Two of the protein phosphatases are shown to have subunit structures. All of the protein phosphatase activities described were able to dephosphorylate sites on proteins that had previously been phosphorylated by the cAMP-dependent protein kinase. Therefore it may be that these phosphatases function in vivo to antagonize the action of the cAMP-dependent protein kinase. Various pnpp phosphatase activities, varying in size, cation requirements and pH optima are described. Since many pnpp phosphatases also function with protein substrates it is possible that these activities represent protein phosphatases that function with phosphoproteins that have not been tested.
Evidence for the existence of protein kinase C in Dictyostelium is presented. A histone protein kinase was found in Dictyostelium extracts that eluted from DE52 cellulose at the same salt concentration that rabbit brain protein kinase C did. In addition in one experiment clear dependency on calcium and phospholipids is shown.
The purification to homogeneity, and characterization of a low molecular weight autophosphorylating protein kinase, pp20, is described. This protein kinase is a major phosphorylated protein and in addition represents at least 0.03% of the total cellular protein. The autophosphorylation reaction can be inhibited by EDTA but not by EGTA. Following inhibition by EDTA, autophosphorylation can be reactivated by the addition of Mn²⁺, Mg²⁺, or Ca²⁺. Western blotting evidence is presented that shows cross reactivity of pp20 and an 85 KDa protein that may possess casein kinase activity.Ph. D.
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Baldridge, Anthony Owen. "Synthesis, photophysics, and application of fluorescent protein chromophore analogs." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/44744.
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The green fluorescent protein chromophore exhibits remarkably different properties upon removal from the protective beta-barrel. This work focuses on the synthesis of these chromophores as wells studying the photophysics as to why they readily deactivate. Following these initial discoveries, these chromophores can be applied to many different environments providing a fluorescence "turn-on" and thus proving to be applicable in a number of different environments and fields.
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Walker, Douglas Gordon. "Characterization of immediate-early and early proteins of murine cytomegalovirus synthesized in permissive and nonpermissive cells." Thesis, University of British Columbia, 1985. http://hdl.handle.net/2429/25985.
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The gene products produced by murine cytomegalovirus (MCMV) in infected cells prior to viral DNA synthesis are believed to control the interaction of the virus with the cells, determining whether a permissive infection results, with virus replication, or whether further virus gene expression is inhibited, resulting in a latent or abortive infection. The aim of this study was to characterize the early viral gene products that are produced in permissive and nonpermissive cells.
The proteins produced in 3T3-L1 cells, permissively infected with MCMV, during the first six hours of infection (the period prior to viral DNA replication) were characterized by polyacrylamide gel electrophoresis. Ten of the proteins were classified as immediate-early (IE) and seven as early according to their time of synthesis and also according to their synthesis in the presence of actinomycin D following the reversal of a cycloheximide mediated block in protein synthesis. The estimated molecular weights ranged from 28K - 100K. The synthesis of a dominant IE protein of 100K was significantly increased, after the reversal of a cycloheximide block, compared to unenhanced conditions. The synthesis of two other major IE proteins of 96K and 89K were also significantly enhanced by this treatment. The 100K and 89K proteins partitioned with the nuclear, cytoplasmic and cytoskeletal fractions, while the 96K protein partitioned more strongly with the nuclei. These proteins were phosphorylated. The other IE proteins were synthesized in lesser amounts. The major early proteins, which had molecular weights of 39K and 36K, were also phosphorylated and were exclusively nucleus-associated. A number of the IE and early proteins had affinity for native and denatured DNA-cellulose.
The same major IE and early proteins were identified in nonpermissively infected J774A.1 macrophage cells. Although 0.6% of these cells became permissively infected with MCMV and the rest appeared to be nonpermissively infected, viral DNA and late protein synthesis was not detected. The major difference between the proteins produced in 3T3-L1 cells and J774A.1 cells was the affinity of the 96K protein for denatured DNA-cellulose, which was only observed when the protein was synthesized in J774A.1 cells.
The main IE and early MCMV induced proteins were also synthesized in nonpermissively infected human fibroblast cells. The only difference between the proteins produced in these cells and 3T3-L1 cells was that the 100K IE protein appeared to have a greater nuclear-affinity, when produced in the human fibroblasts, than was found when synthesized in infected 3T3-L1 cells.
In conclusion, a larger number of IE and early MCMV-induced proteins were identified in infected cells than had been previously characterized. There was no evidence of restricted MCMV gene expression occurring in two different cell types that were nonpermissively infected. This appeared to indicate that, in the nonpermissive experiments described, MCMV replication was inhibited at the stage of viral DNA synthesis.Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
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Bose, Christopher Cyril. "Peptide synthesis, protein folding and stability." Thesis, University of Leicester, 2000. http://hdl.handle.net/2381/29644.
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The aim was to evaluate the effects of backbone substituents on the stability of protein domains. The substituents used were a variety of peptidomimetics and uncommon amino acids. The highly constrained substituents were introduced by total chemical synthesis using solid phase peptide synthesis techniques. Protein G B2 domain analogues containing 6-amino hexanoic acid, bisthiazolidine dipeptide (BTD), aminoisobutyric acid, D-proline and N-methyl alanine were made using (O- (7-Azahydroxybenzotriaz-1-yl)-1,1,3,3,- tetramethyl uronium hexafluorophosphate (HATU) as a coupling agent for hindered residues. The stabilities, relative to wildtype, of nine synthetic analogues were analysed by denaturant unfolding, circular dichroism and differential scanning calorimetry. Binding to hA33 Fab was analysed using surface plasmon resonance methods. The wildtype sequence had an affinity for Fab of 35M. The mutant K15d-P T16N-MeA was 2kJmol-1 more stable than wildtype and had an affinity for Fab of 280M. The mutant Q37Aib was just as stable as wildtype and had an affinity for Fab of 250M. It is now possible to make analogues of protein domains with any amino acid residue, no matter how hindered, anywhere in the sequence. The introduction of restrained turn mimetics perturbs the folded state but does not destroy it. The introduction of uncommon side chains can make domains more stable than the wildtype structure and maintain the ability to bind antibodies to the wildtype. Structure/activity studies on small proteins can now be extended to use any uncommon amino acid in addition to the naturally occurring ones.
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Lovmar, Martin. "Macrolide Antibiotics in Bacterial Protein Synthesis." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6009.
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Tang, Norina Mei Ngon. "Regulation of protein synthesis and induction of oncogenesis by a cellular protein kinase inhibitor /." Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/11501.
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Zhang, Yinfeng, and 张银凤. "Protein chemical synthesis by serine and threonine ligation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/202359.
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Landmark advances in the field of synthetic protein chemistry have enabled the preparation of complex, homogeneous proteins, including those that carry specific posttranslational modifications (PTMs). In addition, chemical synthesis will allow one to incorporate unnatural elements to generate new biologics with altered properties and functions. Native chemical ligation (NCL) is a milestone in the chemical synthesis of proteins (Kent et al., Science, 1994, 266, 776-779), in which a C-terminal peptide thioester and an N-terminal cysteine (Cys)-containing peptide-both in side-chain unprotected forms-are selectively coupled to generate a natural peptidic linkage at the site of ligation. This method requires a cysteine at the optimal convergent ligation site. However, Cys is one of the least abundant amino acids in natural proteins. Therefore, the development of new ligation methods at other amino acids will be necessary and important in this regard.
Along these lines, our laboratory has developed a novel thiol-independent approach-serine/threonine ligation (STL). It uses the N-terminal serine or threonine of a peptide segment to chemoselectively react with another peptide segment with a C-terminal salicylaldehyde ester to form an N,O-benzylidene acetal linked product, followed by acidolysis to afford the final product at the natural Ser/Thr site. To extend the application of STL in chemical protein synthesis, we have developed a robust method for the preparation of peptide salicylaldehyde esters via Fmoc-based solid phase peptide synthesis. Furthermore, we have successfully applied this ligation method in the convergent synthesis of peptide drugs of significant therapeutic importance, including Teriparatide (Forteo), Corticorelin (oCRH), Exenatide (Byetta) and Tesamorelin (hGHRH). Of significance, we have demonstrated the effectiveness of our STL in the assembly of a more complex target of biological interest: human erythrocyte acylphosphatase (~ 11 kDa).
In summary, we have developed a new serine/threonine ligation, which can be effectively used to synthesize peptides and proteins. As there are countless serine and threonine residues in natural proteins, particularly those carrying posttranslational modifications, this method is anticipated to offer new opportunities in synthetic protein chemistry and chemical biology.published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy
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Zhang, Guangtao. "Design, synthesis, and evaluation of cholera toxin inhibitors and [alpha]-helix mimetics of dormancy survival regulator /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/8485.
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Neubauer, Cajetan Simon Johannes. "Structural aspects of quality control in protein synthesis." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609457.
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Bruell, Christian M. "Mechanism of protein synthesis in Mycobacterium smegmatis /." Zürich : ETH, 2008. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17733.
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Johansson, Magnus. "Rate and Accuracy of Bacterial Protein Synthesis." Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-171040.
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High levels of accuracy in transcription, aminoacylation of tRNA, and mRNA translation are essential for all life forms. However, high accuracy also necessarily means large energy dissipation and slow kinetics. Therefore, in vivo there is a fine tuned balance between rate and accuracy of key chemical reactions. We have shown that in our optimized in vitro bacterial protein synthesis system we have in vivo compatible rate and accuracy of ribosomal protein elongation. Our measurements of the temperature and the pH dependence of peptide bond formation with native substrates also suggest that the chemical step of peptidyl transfer, rather than tRNA accommodation, limits the rate of peptide bond formation. This work has made it possible to study ribosomal peptidyl transfer with native substrates. Furthermore, we have developed a general theoretical model for the rate-accuracy trade-off in enzymatic reactions. When considering this trade-off for protein synthesis in the context of the living bacterial cell, where cognate aa-tRNAs compete for ribosome binding with an excess of non-cognate aa-tRNAs, the model predicts an accuracy optimum where the inhibitory effect of non-cognate substrate binding and the efficiency loss due to high discard rate of cognate aa-tRNAs are minimized. However, these results also show that commonly used biochemical systems for protein synthesis studies operate at exceptionally suboptimal conditions. This makes it difficult, if not impossible, to relate the biochemical data to protein synthesis in the living cell. To validate our theoretical model we developed a method, based on variation of the concentration of Mg2+ ions in the buffer, to study the rate-accuracy trade-off of bacterial protein synthesis in vitro. We found a linear trade-off between rate and accuracy of tRNA selection on the ribosome, from which we could estimate the maximal accuracy. Exploiting this method for a complete set of single-mismatch readings by one tRNA species, we found simple patterns of genetic code reading, where the accuracy was highest for the second and lowest for the third codon position. The results bridge the gap between in vivo and in vitro protein synthesis and allow calibration of our test tube conditions to those of the living cell.
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Bhutia, Pema choden. "Accuracy of TransferRNA Selection in Protein synthesis." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-162811.
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ACCURACY OF TRANSFER RNA SELECTION IN PROTEIN SY The ribosome is a rapid magnificent molecular machine that plays an important role in proteinsynthesis and it consists of RNA and protein. The 70S bacterial ribosome comprises twosubunits, 30S and 50S. The 30S small subunit of the bacterial ribosome contains a protein calledS12, encoded by the rpsL gene. The function of this S12 protein is to help arrange the mRNAcorrectly to the ribosome and to interact with transfer RNA (tRNA) to initiate translation.Mutations in the rpsL gene generate phenotypes like resistance, dependence or pseudodependenceto the antibiotic streptomycin in bacteria. It is believed that mutations in the rpsLgene can increase the accuracy of tRNA selection in protein synthesis.The ribosome conducts the selection of tRNA in two steps: the initial selection and theproofreading step. During these multiple steps, the ribosome chooses the cognate aminoacyltRNAsin a ternary complex with EF-Tu and GTP and accommodates in the A site of ribosome.Therefore, the accuracy of the ribosome in selection of cognate aminoacyl-tRNA is crucial for the production of functional polypeptide sequences. Here, three different Escherichia coli strains; wild type MG1655, streptomycin restrictive (SmR) strain res222, and a streptomycin pseudo-dependent (SmP) strain w3110 are used, for studying the accuracy of tRNA selection inprotein synthesis. The mutant SmR shows hyper-accurate phenotype, which means, it has lowerpeptide bond formation efficiency and higher accuracy than the wild type. SmP shows pseudodependentto streptomycin phenotype which means it has higher peptide bond formation efficiency in the presence of antibiotic streptomycin. I have estimated the accuracy of tRNA selection in protein synthesis with enzyme kinetics. The kinetics data of these experiments display that mutant streptomycin restrictive is hyper-accurate and lower peptide bond formation efficiency than the wild type. SmP for the near cognate reaction in presence of antibiotic streptomycin has higher peptide bond formation efficiency than the SmP in absence of antibiotic streptomycin. SmP in presence antibiotic streptomycin has lower accuracy than the SmP in absence of antibiotic streptomycin.
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Khan, Mohammad Farhan. "Robustness and responsiveness in eukaryotic protein synthesis." Thesis, University of Kent, 2017. https://kar.kent.ac.uk/63887/.
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Phosphorylation of eukaryotic translation initiation factor 2 (eIF2) is one of the best studied and most widely used means for regulating protein synthesis activity in eukaryotic cells. Control through eIF2 is exerted by its phosphorylation, which disrupts the guanidine exchange cycle that is required for every initiation event, and thereby inhibits translation. The eIF2 pathway regulates protein synthesis in response to stresses, viral infections, and nutrient depletion, among others. We present analyses of an ordinary differential equation-based model of this pathway, which aim to identify its principal robustness and stability conferring features using linear control theory. The eIF2 pathway can respond sensitively, appropriately and in a timely fashion to some changes in the environment of the cell, while being robust and unresponsive to other types of change. Neither the way in which appropriate responses are achieved (responsiveness), nor how inappropriate responses are avoided (robustness), is currently well understood. Our analyses indicate that, within eIF2 dependent regulatory model the robustness do not arising from the properties of any one individual pathway species rather is a distributed property. On the other hand, stability lies in the structure of the model that is damaging the structure produces major alterations in the stability. Further it is observed that, key non-linearities within the system helps in maintaining transient behaviour and removal or linearisation of key non-linearities can generate undesirable results such as negative cellular concentration. Our analyses also indicate existence of natural sliding surface within the eIF2 dependent regulatory system that helps the system in counteracting uncertainties lies in the input channel. Further, the role of uncharged transfer ribonucleic acid (tRNA) and protein kinase R (PKR) signalling on general translation rate as well as on phosporylation of eIF2-alpha is also investigated by extrapolating the proposed computational yeast model to the case of mammalian cells. It is observed that PKR is compensating the loss of general control nonderepressible 2 (GCN2) and maintaining levels of phosphorylated eIF2-alpha in tumours, while signalling strength of uncharged tRNA is responsible for delivering statistically significant difference in the ratio of phosphorylated eIF2 and alpha-subunit of eIF2 for mixed background and C57BL6 sarcomas.
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Muir, Thomas W. "Synthesis and conformational studies of a protein." Thesis, University of Edinburgh, 1992. http://hdl.handle.net/1842/15456.
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An investigation into the conformational stability of the small globular protein ubiquitin (Ub) is described. Solid phase peptide synthesis has been successfully applied to the construction of both mammalian ubiquitin, as well as to a ubiquitin analogue (Ubdes-Core) lacking the protein's pronounced hydrophobic core. In each case the synthetic protein was purified to homogeneity using a combination of gel filtration, dialysis and ion exchange chromatography. Synthetic ubiquitin was found to be fully active in a ubiquitin specific in vitro protein conjugation assay, whereas Ubdes-Core was found to be completely inactive. The structure of these two proteins has been studied in some detail. Synthetic ubiquitin possesses an identical crystal and NMR derived solution structure to its natural counterpart. Removal of the hydrophobic core from ubiquitin results in a substantial loss in conformational stability (3.7 kcal mol-1) and consequently Ubdes-Core is unable to adopt the ubiquitin native fold. This leads to the conclusions that hydrogen bonding contributions are not in themselves sufficient to stabilize the native conformation of ubiquitin. The preparation of a 128 residue precursor protein comprising ubiquitin fused to a C-terminal extension protein (CE-52) is presented along with the CEP52 fragment alone. The compounds were prepared by solid phase peptide synthesis and purified using a combination of gel filtration, ion exchange and HPLC. Two-dimensional NMR analysis of the UbCEP52 precursor revealed, significantly, that it possesses no well defined tertiary structure. However, the protein was found to be processed in vitro to mature ubiquitin and CEP52. A possible novel function of ubiquitin in unfolded C-terminally fused conujugates is suggested.
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Cottam-Howarth, Barry. "Towards PNA directed chemical total protein synthesis." Thesis, University of Leeds, 2007. http://etheses.whiterose.ac.uk/21098/.
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This thesis is concerned wi.th the development of a novel methodology for the chemical synthesis of proteins. The methodology is expected to overcome limitations of current techniques for chemical synthesis of proteins that restrict the size of the target molecule to less than 150 amino acids. It is intended that the use of a DNA analogue, PNA, as a directing group will allow different short polypeptides to self-assemble into a desired target protein. The first chapter is an overview of literature concerning templated synthesis and the applications of PNA. Examples of simple mctal-templated synthesis are discussed however there is a particular focus on the work of David Liu and his DNA-templated synthesis. The uses of PNA in a number of diverse applications are presented along with brief comparative descriptions of PNA and DNA. A brief discussion of current methodologies for protein synthesis is then used to set the context for the project aims and a description of the proposed methodology. The second chapter details the effort made in modifying literature procedures to successfully develop the robust and reproduCible cqemistry required for multi-gram synthesis ofPNA monomers. The third chapter briefly discusses the computer-aided design of a pair of linker molecules that act as a bridge between polypeptides and their PNA directing groups. The challenging synthesis of these linker molecules is detailed and synthetic routes for the multi-gram synthesis of both are given. The fourth chapter charts the development of the solid-supported chemistry used to construct PNA-peptide chimeras with close attention to the challenges encountered with incorporating the linker molecules. Of the two chimera targets required. to examine PNA-directcd ligation, a multi-gram preparative synthetic route is presented for one chimera. A route that provides analytical quantities of the complementary chimera is also discussed. The fifth chapter presents the experimental procedures used for the synthesis of the compounds involved in this project. Analytical data and the characterisation of compounds are detailed for each compound where possible.
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Varanda, Ana Sofia Paulo. "Aberrant protein synthesis in human HEK293FT cells." Master's thesis, Universidade de Aveiro, 2011. http://hdl.handle.net/10773/7287.
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Mestrado em Biologia Aplicada - Biologia Molecular CelularProtein synthesis is tightly regulated and fidelity in this process is essential for maintenance of essential cellular functions. Under normal physiological conditions errors can occur, at frequencies around 10-4 errors per codon decoded. This level of mistranslation can be tolerated by cells. When the frequencies of errors increase mechanisms of protein quality control can fail leading to accumulation of misfolded proteins, which are more prone to form toxic aggregates, can compromise cell viability, disrupt cellular homeostasis and lead to the development of diseases. In order to elucidate how cells respond to mistranslation and understand how protein accumulation can cause disease and cell degeneration, we exposed human HEK293FT cells to canavanine and azetidine-2-carboxylic acid (AZC) which are analogues of arginine and proline, respectively. Their misincorporation into proteins leads to erroneous protein synthesis, misfolding and likely to protein aggregation. Such mistranslation event decreased slightly cellular viability and increased the number of cells arrested in G2/M and S phases of cell cycle. In HEK293FT cells was detected an increase in proteins conjugated with ubiquitin, without altering proteasome activity, which may indicate that at this level of mistranslation, protein quality control mechanisms are active to counteract the formation of protein aggregates. Transcriptional analysis showed that down-regulated genes were mainly associated with extracellular matrix and cell adhesion and up-regulated genes were involved in negative regulation of transcription and response to unfolded proteins. This study provides new insights into the response of human cells to mistranslation by giving a global analysis of transcriptional alterations that occur in response to proteotoxic stress. HEK293FT cells can be a good model to understand the molecular basis of human diseases caused by mRNA mistranslation.
A síntese proteica é um mecanismo sujeito a uma apertada regulação, pois a manutenção de funções celulares essenciais está dependente da fidelidade deste processo. Em condições fisiológicas normais podem ocorrer erros, a uma frequência de aproximadamente 10-4 erros por codão descodificado. Este nível de erro é tolerado pelas células, mas quando a frequência destes aumenta, os mecanismos de controlo de qualidade das proteínas podem falhar levando à acumulação de proteínas aberrantes (misfolded) que tendem a formar agregados tóxicos, podem comprometer a viabilidade celular, alterar a homeostasia e levar ao desenvolvimento de doenças. Com o objectivo de elucidar os mecanismos de resposta à acumulação de erros durante a síntese proteica e entender como o aumento de proteínas aberrantes pode levar ao desenvolvimento de doenças e degeneração celular, foram utilizadas células HEK293FT expostas a canavanina e azetidine-2- carboxylic acid (AZC), análogos da arginina e prolina respectivamente. A incorporação destes análogos de aminoácidos leva a uma síntese proteica aberrante e origina proteínas que tendem a agregar e ter consequências tóxicas para as células. No nosso estudo observámos que a incorporação de análogos de aminoácidos levou a uma diminuição ligeira da viabilidade celular, assim como levou ao aumento do número de células nas fases G2/M e S do ciclo celular. Também foi detectado um aumento de proteínas conjugadas com ubiquitina, não se observando alteração na actividade do proteasoma. Isto pode indicar que a este nível de erro a que as células estão sujeitas, os mecanismos de controlo de qualidade de proteínas estão a ser activados de forma a evitarem a agregação proteica. Através das análises ao transcriptoma observou-se que os genes cuja expressão diminuiu estão relacionados com a matriz extracelular e adesão celular e os genes cuja expressão aumentou, estão envolvidos na regulação negativa da transcrição e na resposta a proteínas aberrantes. Este estudo permitiu adquirir novos conhecimentos acerca da resposta celular à incorporação de erros durante a síntese proteica, através da análise de alterações transcripcionais causadas pelo stress proteotóxico. As células HEK293FT são um bom modelo de estudo para compreender as bases moleculares de doenças humanas originadas por uma síntese proteica aberrante.
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Hu, Yaogang. "Design and Synthesis of Bioactive Peptidomimetics." Scholar Commons, 2015. https://scholarcommons.usf.edu/etd/5504.
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Protein-Protein Interactions (PPIs) play a very important role in biological functions and therefore the inhibition of specific Protein-Protein Interactions has a huge therapeutic value. The most successful small molecular PPIs inhibitors do not fit with the prevalent `Rule of Five' drug profile. To overcome the disadvantages of small molecular PPIs inhibitors, peptide based PPIs inhibitors were developed. Herein we describe the development of a new class of peptidomimetics AA-peptides. The AApeptides were designed based on chiral PNA backbone. Substitution of nucleobases yields AApeptides that are resistant to proteolysis and capable of mimicking peptides. Two types of AApeptides were discussed in this dissertation "α-AApeptides" and "γ-AApeptides". The AApeptides were shown to disrupt p53/MDM2 protein-protein interaction and tomimic fMLF tripeptide to target G protein-coupled formyl peptide receptors (FPRs). Moreover, the lipidated α-AApeptides can mimic the structure and function of natural antimicrobial lipopeptides and show broad-spectrum activity against both Gram-positive and Gram-negative bacteria. Lastly I have designed and synthesized a serials of phosphopeptides to disrupt cancer related STAT3-STAT3 dimerization.
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Malmgren, Lisa M. "Using in situ click chemistry to modulate protein-protein interactions : Bcl-XL as a case study." [Tampa, Fla.] : University of South Florida, 2007. http://purl.fcla.edu/usf/dc/et/SFE0002233.
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Scorer, Carol Amanda. "The accuracy of foreign protein translation by Escherichia coli." Thesis, Open University, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.329342.
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Renberg, Björn. "Fluorescence-based ligand assays for protein detection using affibody affinity proteins." Doctoral thesis, KTH, Skolan för bioteknologi (BIO), 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-3936.
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The detection and quantification of biomolecules, and proteins in particular, are of great interest since these molecules are of fundamental importance to our well-being. Body fluids, as for instance human blood, are well suited for sampling of protein levels. However, the complexity of the fluids and the low abundance of many of the interesting biomolecules makes detection and quantification difficult. This has spurred an interest into the development of many protein detection methods, and of these, ligand assays have proven particularly suitable. In this thesis, different types of ligand assays for protein detection have been developed using affibody molecules as ligands. In a first study, a homogeneous competitive detection assay was investigated, based on antiidiotypic affibody molecule pairs and fluorescence resonance energy transfer (FRET) as reporting system. The individual members of two anti-idiotypic affibody pairs, each consisting of a target binding (idiotypic) and an anti-idiotypic affibody ligand, were labeled with a donor fluorophore and an acceptor fluorophore, respectively. Incubation with the two target proteins IgA and Taq DNA polymerase resulted in a concentration dependent decrease in the FRET signal, allowing for target protein detection and quantification. For Taq DNA polymerase, detection in 25% human plasma was also possible in the same concentration span as in buffer. In a second study, a homogeneous, non-competitive detection system was described. Affibody molecules of 58 amino acids directed against IgA and IgG were produced with chemical synthesis, and two fluorophores capable of FRET were site-specifically introduced. Binding of target protein induced a concentration-dependent change in the relative emission of the two fluorophores, which formed the basis for the detection system. In two studies, affibody molecules were evaluated and shown to function well as capture ligands on microarrays. Synthetic affibody molecules directed against Taq DNA polymerase and IgA were modified by the introduction of immobilization tags. Specific immobilization via a C-terminal cysteine or a biotin moiety, or random immobilization via amino groups, were studied in protein microarray experiments and SPR-based biosensor studies. The experiments showed that all immobilization chemistries resulted in functional capture molecules. A short spacer was also introduced, situated between the affibody and the cysteine and biotin moieties, which was shown to improve binding for all constructs. Multidomain affibody constructs of up to four N- to C-terminally linked domains were shown to increase the amount of bound target, compared to monomeric affibody ligands. Six dimeric affibody constructs directed against IgA, IgG, IgE, Taq DNA polymerase, TNF-α and insulin, respectively, showed low limits of detections for their targets and little or no cross-reactivity with the other target proteins. Dimeric affibody molecules directed against IgA and TNF-α were also shown to function in a sandwich format with antibodies for detection of targets in buffer and in human serum and plasma. Successful discrimination between normal and IgA-deficient sera showed that affibody molecules could be used for specific detection of protein in highly complex backgrounds on microarrays.QC 20100916
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Takahshi, Naoko. "Flagella synthesis in Rhodobacter sphaeroides WS8." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259820.
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Huang, Brandon Pei Han. "The regulation of protein synthesis in adult rat cardiomyocytes." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/976.
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Protein synthesis (mRNA) is tightly regulated under numerous conditions in cardiomyocytes. It can be activated by hormones such as insulin and also by other agents such as phenylephrine (PE) that activates hypertrophy in the heart. Cardiac hypertrophy involves an increase in the muscle mass of the heart, principally in the left ventricular muscle, and the increase is due to enlarged cell size, not increased cell number. A pivotal element of cardiac hypertrophy is an elevation in the rates of protein synthesis, which drives the increase in cell size causing hypertrophy. Unfortunately, we currently lack the understanding of the basic mechanisms that drives hyperactivated protein synthesis. Cardiac hypertrophy is clinically important because it is a major risk factor for heart failure. It initially serves as an adaptive response to increase cardiac output in response to higher demand, but ultimately leads to deterioration of contractility of the heart if hypertrophy is sustained. The main goal of this research project is to understand how hypertrophic agents, such as phenylephrine (PE), activate protein synthesis using adult rat ventricular cardiomyocytes as a model. Specifically, this study focuses on how the translational initiation is controlled by upstream signalling pathways.
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Knight, Ashley D. "Is Leucine Intake Associate with Enhanced Muscle Protein Synthesis and Attenuated Muscle Protein Breakdown?" Digital Archive @ GSU, 2013. http://digitalarchive.gsu.edu/nutrition_theses/45.
Full textAbstract:
Is Supplemental Leucine Intake Associated with Enhanced Post Exercise Muscle Protein Synthesis and Attenuated Muscle Protein Breakdown?
Knight AD, Benardot D, Thompson W, and Henes ST
Introduction: The role of individual amino acids on protein synthesis and their impact on physical performance is of high importance to athletes and to those studying the science of sports nutrition. Leucine, one of three branched-chain amino acids, is a frequently researched amino acid because of its potential stimulatory effect on muscle protein synthesis (MPS) following exercise in humans.
Purpose: Although there have been many studies conducted on leucine’s muscle stimulatory effect, questions remain as to the efficacy and feasibility of leucine as an MPS catalyst. Contributing to these questions are the widely varied dosing and timing strategies that different researchers have employed. It is the purpose of this thesis, therefore, to assess the differences in study protocols and shed light on the potential effectiveness on leucine as a MPS stimulator. Central to this issue is whether supplemental leucine intake is associated with enhanced post exercise MPS and, if so, what associated factors, including timing and level of intake, are most likely to influence this effect.
Methods: A comprehensive review of the literature on leucine and its effect on MPS was performed. Studies were organized into similar topics, with an assessment and summary of effect produced for each topic area. A general conclusion was made that was based on the summary of each topic area.
Results: Leucine is involved in protein metabolism regulation through its role in stimulating the mammalian target of rapamycin (mTOR) signaling cascade and by indicating energy and amino acid availability. It functions to initiate MPS and decrease muscle protein breakdown by downregulating the ubiquitin-proteasome system, lysosomal activity, and/or increasing circulating insulin.
Conclusions: Supplementation with the amino acid leucine effectively enhances MPS and attenuates muscle protein degradation in humans following bouts of physical exertion. Leucine intake in amounts greater than that found in ~20g whole protein saturates MPS and increases leucine oxidation. For this reason, an upper limit of leucine intake should be established. While leucine successfully increases MPS, it remains unclear whether this translates to enhanced physical performance, an area that requires more studies to be conducted.
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Wood, Wendy. "The role of individual domains of eIF4G in translation initiation." Thesis, University of Sussex, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322906.
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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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Park, Chihyo. "Combinatorial design and synthesis of peptidomimics and small molecules for protein-protein interactions." Texas A&M University, 2006. http://hdl.handle.net/1969.1/4692.
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The solid phase combinatorial method is an excellent tool for the modulation of
protein-protein interactions through focused library generations. Nucleophilic aromatic
substitution reactions with an iodinated template on solid phase has opened a door for
easy and pure libraries of 13-22 membered medium and macrocyclic peptidomimetics.
These peptide mimics showed promising activities for tyrosine kinase receptors.
Iodine functionality can then be used to modify the products, on the resin, via
Sonogashira and Suzuki couplings and presumably through other organometallic
catalysis. The coupled products can have conformational biases that differ from the
iodinated macrocycles. These coupling reactions also provide a means to introduce
additional pharmacophores and to adjust the solubilities of the products.
The fluorinated template also gave libraries of cyclic peptidomimetics on solid phase
in good yields and purities. These libraries have improved water solubility over the
iodinated libraries. The 3-fluorinated template yielded better results than the 5-
fluorinated template. Some compounds showed biological activities in cell survival
assays providing strong support of our approach to mimic external ò-turn sequences in
target proteins.
Intrasite dimerization with 1,5-hexadiyne gave a homodimer as a byproduct. Solidphase
synthesis of bivalent turn mimics with fluorescent tags has been demonstrated.
The key feature of this synthetic route is that homo- and hetero-dimers can be formed
chemoselectively from unprotected monomeric precursors. The dimerization reaction is
very mild and versatile, as only potassium carbonate is required to affect the coupling.
Solution phase library synthesis of small molecule mimics is presented. Some
monomers of full sequence mimics have been prepared to afford dimer generations. Theses monomers were combined with linker handles to afford diverse length of dimers.
Final combination of monomers to make bivalent compounds is in progress.
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Oddy, V. H. "Muscle protein metabolism : Measurement and manipulation in lambs." Thesis, University of Cambridge, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382662.
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Spjut, Sara. "Glycoconjugates synthesis and investigation of carbohydrate-protein interactions /." Doctoral thesis, Umeå : Kemiska institutionen, Umeå universitet, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-33841.
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Wikström, Malin. "Synthesis and protein curing abilities of membrane glycolipids." Doctoral thesis, Stockholm University, Department of Biochemistry and Biophysics, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-1361.
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There are many types of membrane lipids throughout Nature. Still little is known about synthesizing pathways and how different lipids affect the embedded membrane proteins. The most common lipids are glycolipids since they dominate plant green tissue. Glycolipids also exist in mammal cells as well as in most Gram-positive bacteria. Glycosyltransferases (GTs) catalyze the final enzymatic steps for these glycolipids. In the bacteria Acholeplasma laidlawii and Streptococcus pneumonie and in the plant Arabidopsis thaliana, GTs for mono-/di-glycosyl-diacylglycerol (-DAG) are suggested to be regulated to keep a certain membrane curvature close to a bilayer/nonbilayer phase transition. The monoglycosylDAGs are nonbilayer-prone with small headgroups, hence by themselves they will not form bilayer structures.
Here we have determined the genes encoding the main glycolipids of A. laidlawii and S. pneumonie. We have also shown that these GTs belong to a large enzyme group widely spread in Nature, and that all four enzymes are differently regulated by membrane lipids. The importance of different lipid properties were traced in a lipid mutant of Escherichia coli lacking the major (75 %), nonbilayer-prone/zwitterionic, lipid phosphatidylethanolamine. Introducing the genes for the GTs of A. laidlawii and two analogous genes from A. thaliana yielded new strains containing 50 percent of glyco-DAG lipids. The monoglyco-DAG strains contain significant amounts of nonbilayer-prone lipids while the diglyco-DAG strains contain no such lipids. Comparing these new strains for viability and the state of membrane-associated functions made it possible to connect different functions to certain lipid properties. In summary, a low surface charge density of anionic lipids is important in E.coli membranes, but this fails to be supportive if the diluting species have a too large headgroup. This indicates that a certain magnitude of the curvature stress is crucial for the membrane bilayer in vivo.
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Sienna, Nancy. "Regulation of ribosomal protein L32 synthesis by hormones." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0027/NQ51047.pdf.
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Wikström, Malin. "Synthesis and protein curing abilities of membrane glycolipids /." Stockholm : Department of Biochemistry and Biophysics, Stockholm University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-1361.
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Zamiri, Maryam. "Synthesis of protein arginine N-methyltransferase 6 inhibitors." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43808.
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Protein arginine N-methyltransferases (PRMTs) are pertinent targets for drug discovery as their dysfunction is associated with a number of diseases such as cancers, cardiovascular diseases and viral pathogenesis. The precise role of PRMTs in the initiation, development, or progression of diseases is not known yet.
Due to association of PRMT1 and 4 with transcriptional activation, the main focus of inhibitor discovery has been on these two enzymes. On the other hand, the goal of this study is to find a PRMT6 specific inhibitor. PRMT6 methylates DNA polymerase β, histones H3 and H4 and HIV proteins: Rev and Tat.
PRMT6 uses S-adenosyl-L-methionine (AdoMet) as the “methyl group” source. AdoMet fits into a distinct conserved binding site in the enzyme, which is located adjacent to the protein substrate/catalytic site such that its S⁺-Me motif is correctly positioned with respect to the substrate arginine nitrogen atom that undergoes methylation.
Based on crystallography data for PRMT1, the purine C8 center in AdoMet is in close proximity to the methionine sulfur atom (M166 in PRMT6). As shown by Frankel et al. (Faculty of Pharmaceutical Sciences, UBC), the M166C PRMT6 mutant displays activity. Based upon this observation, we hypothesize that Ado-Met analogues with reactive substituents (e.g., CHO) at C8 position of adenine ring will form a covalent bond with the proximal Cys SH group in M166C PRMT6. This validates our further hypothesize that in appropriately designed analogues, it will be possible to subsequently detach the sugar and amino acid components of Ado-Met to leave the adenine ring component alone bound to the enzyme. This provides a unique opportunity to explore the “fragment based approach in drug discovery” to design PRMT6 specific inhibitors.
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Hong, Wei. "Design and synthesis of protein arginine methyltransferase inhibitors." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/12835/.
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Biological methylation is defined as the transfer of a methyl group from S-adenosyl-L-methionine(SAM) to one of a wide range of potential acceptors such as DNA, RNA, protein, hormones and neurotransmitters. Protein arginine methylation is a common post-translational modification facilitated by protein arginine methyltransferases(e.g. PRMTI). The roles of these enzymes in vivo are currently poorly understood. The focus of the project is design and synthesis of PRMT inhibitors with the ultimate goal of evaluating their activities in cells. Preliminary work toward the synthesis of S-adenosyl-trifluoromethyl-L-homocystein and adenosyl 5'-[2-(tert-butoxycarbonylamino)ethyl-trifluoro methyl] thiophenium is described. The ternary crystal structure of PRMTI in complex with S-adenoSyl-L-homocystein(eSAH) and an arginine containing peptide (PBD IOR8) was used to design a series of potential bisubstrate inhibitors of PRMTI. The prototypical SAM analogues bearing guanidine group were sought to replace the reactive sulfonium centre with nitrogen. Analogue synthesis proceeded via successive reductive arnination of Y-arnino-Y-deoxyadenosine and deprotection in good overall yields. An alkyne SAM analogue, 5'-[(S-3-amino-3-carboxypropyl)-propargylaminol-5'-deoxyadenosine was prepared, which underwent efficient Cu(1) catalysed Huisgen reaction to yield a triazole derived SAM analogue 5'-[(S-3-amino-3-carboxypropyl)-[I-(2-guanidinoethyl)-IH-1,2,3-triazol-4-yl]methyl-amino]-5'-deoxyadenosine. Preliminary biological evaluation of the compounds by collaborators Professor Steve Ward and Dr Richard Parry at the University of Bath, confirmed that 5'-[(S-3-amino-3-carboxypropyl)- 3-guanidinopropyl-amino]-5'-deoxyadenosine and 5-[(S-3-amino-3-carboxypropyl)-5-guanidinopentyl-amino]-5'-deoxyadenosine are potent inhibitors of PRMTI but not the lysine methyltransferase SET7. A related N-6 modified SAM analogue 5'-[(S-3-amino-3-carboxypropyl)-3-guanidinopropylamino]-5'-deoxy-N6-(lI-azido-3,6,9-trioxaundecane)-amino adenosine bearing an azide tether was developed with the aim of allowing facile introduction of biotin or fluorescent dyes, using either Staudinger ligation, or Cu(1) catalysed Huisgen reaction to provide compounds that can be used for affinity purification of the target protein or study of its localisation in cells respectively. Finally, progress toward a novel, rapid and enantioselective synthesis of the natural product (+)-sinefungin is reported. Key dihydropyridazine intermediates were generated from adenosyl 5'-propaldehyde, commercially available azodicarboxylate derivatives and ester substituted vinyltriphenylphosphonium salt by successful extension of methodology first reported by Ley and co-workers. Deprotection and ring opening of clihydropyridazine compounds was attempted, and unfortunately we were not able to generate (+)-sinefungin, although it is hoped that this route can be developed to achieve this in the future.
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Meiries, Sebastien. "Towards the synthesis of novel protein phosphatase inhibitors." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/641/.
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Protein phosphatases (PPases) are important enzymes which mediate dephosphorylation of proteins in eukaryotic cells. These enzymes are involved in a variety of cellular processes, and disruption of their activity has been shown to be involved in the development of diseases such as cancers and Alzheimer’s. Protein phosphatase inhibitors (PPIs) have been used to regulate the activity of these enzymes, and hence, control the development of the cellular processes related to them. However, the lack of potent and selective readily accessible PPIs is a major obstacle to the understanding of these complex biological pathways, and the development of reliable synthetic routes towards new PPIs has therefore generated a significant amount of interest. Several naturally occurring PPIs possess the “ADDA” residue , which has been shown to be crucial for their inhibitory activity. We would like here to report the synthesis of “ADDA”-isoforms such as enantio-ADDA 1 and enantio-iso-ADDA 2 through a convergent approach taking advantage of cross-metathesis methodology, non-aldol aldol and aza-Claisen rearrangements, as well as β-lactam chemistry. We envisage using our synthetic approaches as a platform to generate a wide range of novel “ADDA”-containing analogues, which might lead to the discovery of potent and selective PPIs, which could be generated in multi-gram scale.
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Bathgate, B. "Plastid protein synthesis during fruit development and ripening." Thesis, University of Nottingham, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.482881.
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Parker, J. E. "Developmental regulation of protein synthesis in Euglena gracilis." Thesis, Swansea University, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.638412.
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The expression of mitochondrial citrate synthase (CS) and the cytosolic enzyme NADP-dependent glutamate dehydrogenase (NADPH-GDH) was examined in Euglena gracilis Klebs strain z Pringsheim in relation to light and glutamate provision. Exposure of dark grown cells to white or red light but not blue light caused a transient increase in CS and NADPH-GDH specific activity over the first 12 hours of regreening. Illumination with white or red light but not blue light also initiated efficient chloroplast development over 72 hours. Provision of L-glutamate as sole nitrogen source caused an increase in the specific activity of both enzymes during the first 48 hours of organotrophic growth. Enhanced enzyme activity was shown to be due to an increase in enzyme protein by immunochemical titration, using monospecific antisera raised against the homogeneously pure proteins. Poly(A)-containing RNA was extracted from regreening and glutamate-grown cells and used to programme a reticulocyte or wheat germ lysate in vitro translation system. The translatable poly(A) RNA profiles remained constant during regreening in white or blue light, and in relation to nitrogen provision. Messenger RNAs encoding CS and NADPH-GDH were detectable at all developmental stages by immunoprecipitation, independent of increases in enzyme synthesis. Results suggest that protein expression operates primarily at a post-transcriptional level in Euglena. In order to study the role of poly(A) RNA abundance in developmental expression, a cDNA library was constructed in E. coli for the isolation of CS and NADPH-GDH cDNAs to probe their target mRNA species. Full length double stranded cDNA was synthesised from Euglena poly(A) RNA, enriched in CS and NADPH-GDH mRNAs by denaturing sucrose gradient fractionation. The cDNA library was screened for CS cDNA with a complementary 17'mer mixed oligodeoxynucleotide probe. Several positive colonies were isolated. The further characterisation of cDNA from two strongly hybridising clones, by Northern-blotting, hybrid-release translation, and SP6-directed in vitro transcription is described. Other positively hybridising clones await further analysis. Heterologous cDNA probes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase (RuBPCase) from pea and Chlamydomonas, and the major light harvesting chlorophyll a/b apoprotein from pea, did not cross-hybridise with Euglena RNA. However, a maize genomic clone encoding the large subunit of RuBPCase was used to measure the relative abundance of large subnit mRNA in Euglena during regreening of cells in white and blue light. Results showed that the lack of a blue light response during proplastid development was apparent at the level of chloroplast gene expression.
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Choy, M. K. "Plastid protein synthesis and plastid-to-nucleus signalling." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597661.
Full textAbstract:
Two plastid-to-nucleus signalling pathways had previously been identified by studies on genomes uncoupled (gun) mutants of Arabidopsis. Five putative gunl-like mutants from a new collection of Arabidopsis gun mutants with a green fluorescent protein (GFP) marker gene under the control of a tobacco RbcS (encoding ribulose-1,5-bisphosphate carboxylase small subunit) promoter were examined further. One of the mutant lines, PR48.2N, showed two-fold higher transcript abundance of nuclear photosynthesis genes, RBCS and LHCB1 (encoding light-harvesting chlorophyll a/b-binding protein 1), compared to wild type with or without treatments of norflurazon or lincomycin. Pigment analysis of PR48.2N seedlings, illuminated for 16 hours after being subjected to various lengths of dark treatment demonstrated that the mutant line accumulated less chlorophyll than wild type after short periods of darkness (2-4 days) but showed an enhanced ability to green after prolonged dark treatments (5-10 days). Consistent with the enhanced greening ability, transcript abundance of nuclear photosynthesis genes was higher and there was more thylakoids membrane in chloroplasts in greened PR48.2N seedlings after prolonged darkness compared to the wild type. Microarray analysis indicated that a group of transcripts encoding seed storage proteins, oleosins and late embryogenesis abundant proteins showed very low abundance in PR48.2N seedlings. The promoter regions of the genes shared some cis-elements possibly involved in regulation by ascisic acid (ABA). However, the ABA content of PR48.2N seedlings was not significantly different to wild type, although the germination of mutant seeds was more sensitive to inhibition by ABA than the wild type.
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Colthurst, David Robert. "A study of protein synthesis in Candida albicans." Thesis, University of Kent, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.280717.
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Stringer, Rebecca Elizabeth. "Endogenous and activated protein synthesis in human neutrophils." Thesis, University of Liverpool, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386847.
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Li, Zhong Qi. "Protein secondary structure mimetics : design, synthesis and evaluation." Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/38780.
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Zhang, Jingji. "Accuracy of mRNA Translation in Bacterial Protein Synthesis." Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-262901.
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Reading of messenger RNA (mRNA) by aminoacyl-tRNAs (aa-tRNAs) on the ribosomes in the bacterial cell occurs with high accuracy. It follows from the physical chemistry of enzymatic reactions that there must be a trade-off between rate and accuracy of initial tRNA selection in protein synthesis: when the current accuracy, the A-value, approaches its maximal possible value, the d-value, the kinetic efficiency of the reaction approaches zero. We have used an in vitro system for mRNA translation with purified E. coli components to estimate the d- and A-values by which aa-tRNAs discriminate between their cognate and near cognate codons displayed in the ribosomal A site. In the case of tRNALys, we verified the prediction of a linear trade-off between kinetic efficiency of cognate codon reading and the accuracy of codon selection. These experiments have been extended to a larger set of tRNAs, including tRNAPhe, tRNAGlu, tRNAHis, tRNACys, tRNAAsp and tRNATyr, and linear efficiency-accuracy trade-off was observed in all cases. Similar to tRNALys, tRNAPhe discriminated with higher accuracy against a particular mismatch in the second than in the first codon position. Remarkably high d-values were observed for tRNAGlu discrimination against a C-C mismatch in the first codon position (70 000) and for tRNAPhe discrimination against an A-G mismatch in the second codon position (79 000). At the same time, we have found a remarkably small d-value (200) for tRNAGlu misreading G in the middle position of the codon (U-G mismatch). Aminoglycoside antibiotics induce large codon reading errors by tRNAs. We have studied the mechanism of aminoglycoside action and found that the drug stabilized aminoacyl-tRNA in a codon selective in relation to a codon non-selective state. This greatly enhanced the probability of near cognate aminoacyl-tRNAs to successfully transcend the initial selection step of the translating ribosome. We showed that Mg2+ ions, in contrast, favour codon non-selective states and thus induce errors in a principally different way than aminoglycosides. We also designed experiments to estimate the overall accuracy of peptide bond formation with, including initial selection accuracy and proofreading of tRNAs after GTP hydrolysis on EF-Tu. Our experiments have now made it possible to calibrate the accuracy of tRNA selection in the test tube to that in the living cells. We will now also be able to investigate the degree to which the accuracy of tRNA selection has been optimized for maximal fitness.