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Ghaddar, Kassem. "Structural analysis of yeast amino acid transporters: substrate binding and substrate-induced endocytosis." Doctoral thesis, Universite Libre de Bruxelles, 2014. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209318.
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Plasma membrane transport proteins play a crucial role in all cells by conferring to the cell surface a selective permeability to a wide range of ions and small molecules. The activity of these transporters is often regulated by controlling their amount at the plasma membrane, via intracellular trafficking. The recent boom in the numbers of crystallized transporters shows that many of them that belong to different functional families with little sequence similarity adopt the same structural fold implying a conserved transport mechanism. These proteins belong to the APC (Amino acid-Polyamine-organoCation) superfamily and their fold is typified by the bacterial leucine transporter LeuT. This LeuT fold is characterized by inverted structural repeats of 5 transmembrane domains that harbor the central substrate-binding site and a pseudo-symmetry axis parallel to the membrane. The yeast Saccharomyces cerevisiae possesses about 16 amino acid permeases (yAAPs) that belong to the APC superfamily and that display various substrate specificity ranges and affinities. Topological, mutational analysis and in silico data indicate that yAAPS adopt the LeuT fold.<p><p>In this work we combined computational modeling and yeast genetics to study substrate binding by yAAPs and the endocytosis of these transporters in response to substrate transport. In the first part of this work, we analyzed the selective recognition of arginine by the yeast specific arginine permease, Can1. We constructed three-dimensional models of Can1 using as a template the recently resolved structure of AdiC, the bacterial arginine:agmatine antiporter, which is also a member of the APC superfamily. By comparison of the binding pockets of Can1 and Lyp1, the yeast specific lysine permease, we identified key residues that are involved in the recognition of the main and side chains of arginine. We first showed that the network of interactions of arginine in Can1 is similar to that of AdiC, and that the selective recognition of arginine is mediated by two residues: Asn 176 and Thr 456. Substituting these residues by their corresponding residues in Lyp1 converted Can1 into a specific lysine permease. In the second part of this work, we studied the regulation of two permeases, Can1 and the yeast general amino acid permease, Gap1. In the presence of their substrates, Gap1 and Can1 undergo ubiquitin-dependent endocytosis and targeting to the vacuolar lumen for degradation. We showed that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. By permease structural modeling, mutagenesis, and kinetic parameter analysis, we showed that Gap1 and Can1 need to switch to an intermediary conformational state and persist a minimal time in this state after binding the substrate to trigger their endocytosis. This down-regulation depends on the Rsp5 ubiquitin ligase and involves the recruitment of arrestin-like adaptors, resulting in the ubiquitylation and endocytosis of the permease.<p><p>Our work shows the importance of the structural analysis of yAAPs to get further insight into the different aspects of their function and regulation. We validate the use of a bacterial APC transporter, AdiC, to construct three-dimensional models of yAAPs that can be used to guide experimental analyses and to provide a molecular framework for data interpretation. Our results contribute to a better understating of the recognition mode of amino acids by their permeases, and the regulation of this transport in response to substrate binding.<br>Doctorat en Sciences<br>info:eu-repo/semantics/nonPublished
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Isogawa, Danya. "Studies on the active site of chitosanase from Paenibacillus fukuinensis and its functional modification for utilizing chitosan." Kyoto University, 2014. http://hdl.handle.net/2433/188769.
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Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(農学)<br>甲第18331号<br>農博第2056号<br>新制||農||1022(附属図書館)<br>学位論文||H26||N4838(農学部図書室)<br>31189<br>京都大学大学院農学研究科応用生命科学専攻<br>(主査)教授 植田 充美, 教授 三上 文三, 教授 小川 順<br>学位規則第4条第1項該当
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Weiser, Armin. "Amino acid substitutions in protein binding." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15962.
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Die Modifizierung von Proteinsequenzen unter anderem durch den Austausch von Aminosäuren ist ein zentraler Aspekt in evolutionären Prozessen. Solche Prozesse ereignen sich nicht nur innerhalb großer Zeiträume und resultieren in der Vielfalt des Lebens, das uns umgibt, sondern sind auch täglich beobachtbar. Diese mikroevolutionären Prozesse bilden eine Grundlage zur Immunabwehr höherer Wirbeltiere und werden durch das humorale Immunsystem organisiert. Im Zuge einer Immunantwort werden Antikörper wiederholt der Diversifizierung durch somatische Hypermutation unterworfen. Ziele dieser Arbeit waren, neue Kenntnisse über die Mikroevolution von Antikörpern während der Immunantwort zu gewinnen und die Beziehung zwischen Aminosäureaustauschen und Affinitätsänderungen zu verstehen. Zu diesem Zweck wurde zunächst gezeigt, dass die SPOT Synthese eine präzise Methode ist, um Signalintensitäten drei verschiedenen Bindungsaffinitätsklassen zuzuordnen. Antikörper-Peptid Bindungsdaten, die aus SPOT Synthese Experimenten generiert wurden, bildeten die Grundlage zur Konstruktion der Substitutionsmatrix AFFI - der ersten Substitutionsmatrix, die ausschließlich auf Bindungsaffinitätsdaten beruht. Diese bildete die Grundlage für die Gewinnung eines reduzierten Aminosäuresatzes. Durch einen theoretischen Ansatz konnte gezeigt werden, dass der reduzierte Aminosäuresatz eine optimale Basis für die Epitopsuche darstellt. Für den Prozess der somatischen Hypermutation und Selektion wurde ein neuer Ansatz präsentiert, um für die Affinitätsreifung relevante Mutationen zu identifizieren. Die Analyse zeigte, dass das Spektrum der selektierten Mutationen viel umfangreicher ist als bisher angenommen wurde. Die Tatsache, dass auch einige stille Mutationen stark bevorzugt werden, deutet darauf hin, dass entweder die intrinsische Mutabilität stark unterschätzt wurde oder, dass Selektion nicht nur auf Affinitätsreifung von Antikörpern basiert sondern auch auf ihrer Expressionsrate.<br>A central task of the evolutionary process is the alteration of amino acid sequences, such as the substitution of one amino acid by another. Not only do these amino acid changes occur gradually over large time scales and result in the variety of life surrounding us, but they also happen daily within an organism. Such alterations take place rapidly for the purposes of defense, which in higher vertebrates, is managed by the humoral immune system. For an effective immune response, antibodies are subjected to a micro-evolutionary process that includes multiple rounds of diversification by somatic hypermutation resulting in increased binding affinity to a particular pathogen. The goal of this work was to provide insights into the microevolution of antibodies during the immune response, including the relationship between amino acid substitutions and binding affinity changes. A preliminary step in this work was to determine the accuracy of the SPOT synthesis technique, which could be shown to be an accurate method for assigning measured signal intensities to three different binding affinity classes. A substitution matrix based on data produced with these binding experiments was constructed and named AFFI. AFFI is the first substitution matrix that is based solely on binding affinity. A theoretical approach has additionally revealed that an AFFI-derived reduced set of amino acids constitutes an optimal basis for epitope searching. For the process of somatic hypermutation and selection, a novel approach to identify mutations relevant to affinity maturation was presented. The analysis revealed that the spectrum of mutations favored by the selection process is much broader than previously thought. The fact that particular silent mutations are strongly favored indicates either that intrinsic mutability has been grossly underestimated, or that selection acts not only on antibody affinity but also on their expression rates.
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Hurley, Eldon Kenneth Jr. "Photolyase: Its Damaged DNA Substrate and Amino Acid Radical Formation During Photorepair." Thesis, Virginia Tech, 2005. http://hdl.handle.net/10919/31084.
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Ultraviolet light damages genomic material by inducing the formation of covalent bonds between adjacent pyrimidines. Cis-syn cyclobutane pyrimidine dimers (CPD)constitute the most abundant primary lesion in DNA. Photolyase, a light-activated enzyme, catalytically repairs these lesions. Although many steps in the photolyase-mediated repair process have been mapped, details of the mechanism remain cryptic. Difference FT-IR spectroscopy was employed to obtain new mechanistic information about photorepair. Purified oligonucleotides, containing a central diuracil, dithymidine, or cyclobutane thymidine dimer, were monitored using vibrational methods. Construction of difference infrared data between undamaged and damaged DNA permitted examination of nucleic acid changes upon formation of the CPD lesion; these experiments indicated that C=O and C-H frequencies can be used as markers for DNA damage. Furthermore, in purified photolyase containing isotopically-labeled aromatic amino acids, we observed that tryptophan residues in photolyase underwent structural changes during photorepair. These data indicate that electron transfer during DNA repair occurs through-bond, and that redox-active, aromatic residues form the pathway for electron transfer.<br>Master of Science
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Li, Hong. "Azotobacter vinelandii Nitrogenase: Multiple Substrate-Reduction Sites and Effects of pH on Substrate Reduction and CO Inhibition." Diss., Virginia Tech, 2002. http://hdl.handle.net/10919/27608.
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Mo-nitrogenase consists of two component proteins, the Fe protein and the MoFe protein. The site of substrate binding and reduction within the Mo-nitrogenase is provided by a metallocluster, the FeMo cofactor, located in the a-subunit of the MoFe protein. The FeMo cofactorâ s polypeptide environment appears to be intimately involved in the delicate control of the MoFe proteinâ s interactions with its substrates and inhibitors (Fisher K et al., 2000c). In this work, the a-subunit 278-serine residue of the MoFe protein was targeted because (i) a serine residue at this position is conserved both in the Mo-nitrogenase from all organisms examined and in the alternative nitrogenases (Dean, DR and Jacobson MR, 1992); (ii) its hydroxyl group hydrogen bonds to the Sg of the a-subunit 275-cysteine residue that directly ligates the FeMo cofactor; and (iii) its proximity to the a-subunit 277-arginine residue, which may be involved in providing the entry/exit route for substrates and products (Shen J et al., 1997).
Altered MoFe proteins of A. vinelandii nitrogenase, with the a278Thr, a278Cys, a278Ala and a278Leu substitutions, were used to study the interactions of H+, C2H2, N2 and CO with the enzyme. All strains, except the a278Leu mutant strain, were Nif+. From measurement of the Km for C2H2 (C2H4 formation) for the altered MoFe proteins, the a278Ala and a278Cys MoFe proteins apparently bind C2H2 similarly to the wild type, whereas the a278Thr and the a278Leu MoFe proteins both have a Km ten-times higher than that of the wild type. Unlike wild type, these last two altered MoFe proteins both produce C2H6. These results suggest that C2H2 binding is affected by substitution at the a-278 position. Moreover, when reducing C2H2, the a278Ala and a278Cys MoFe proteins respond to the inhibitor CO similarly to the wild type, whereas C2H2 reduction catalyzed by the a278Thr MoFe protein is much more sensitive to CO. Under nonsaturating concentrations of CO, the a278Leu MoFe protein catalyzes the reduction of C2H2 with sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity between at least two C2H4-evolving sites. This phenomenon was previously observed with the a277His MoFe protein, in which the a-subunit 277-arginine residue had been substituted (Shen J et al., 1997). Together, these data suggest that the MoFe protein has at least two C2H2-binding sites, one of which may be located near the a277-278 residues and, therefore, most likely on the Fe4S3 sub-cluster of the FeMo cofactor. Like the wild type, N2 is a competitive inhibitor of the reduction of C2H2 by the a278Thr, a278Cys and a278Ala MoFe proteins. Apparently, the binding of N2 in these altered MoFe proteins is similar to that with the wild type MoFe protein, suggesting that the aSer278 residue is not directly involved in N2 binding and reduction. Previous work suggested that both a high-affinity and low-affinity C2H2-binding site were present on the MoFe protein (Davis LC et al., 1979; Christiansen J et al., 2000). Our results are generally consistent with this suggestion.
Currently, there is not much information about the proton donors and how the protons necessary to complete all substrate-to-product transformations are transferred. The dependence of activity on pH (activity-pH profiles) has provided useful information about the nature of the groups involved in proton transfer to the FeMo cofactor and the bound substrate. Approximately bell-shaped activity-pH profiles were seen for all products from catalysis by all the MoFe proteins tested whether under Ar, in the presence of C2H2 as a substrate, or with CO as an inhibitor. The profiles suggested that at least two acid-base groups were required for catalytic activity. The pKa values of the deprotonated group and protonated group were determined from the pH that gave 50% maximum specific activity. These pKa values for the altered a278-substituted MoFe proteins and the a195Gln MoFe protein under various assay atmospheres were compared to those determined for the wild type. It was found that the pKa value of the deprotonated group was not affected by either substitution or changing the assay atmosphere. The wild type MoFe protein has a pKa (about 8.3) for the protonated group under 100% argon that was not affected very much by the substitution by Cys, Ala and Leu, whereas the Thr substitution shifted the pKa to about 8, which was the same as that of the wild type MoFe protein in the presence 10% CO. The pKa values for the protonated group for all the altered MoFe proteins were not changed with the addition of 10% CO. These results suggest that the aSer278 residue, through hydrogen bonding to a direct ligand of the FeMo cofactor, is not one of the acid-base groups required for activity. However, this residue may â fine-tuneâ the pKa of the responsible acid-base group(s) through interaction with the aHis195 residue, which has been suggested (Dilworth MJ et al., 1998; Fisher K et al., 2000b) to be involved in proton transfer to substrates, especially for N2 reduction. The activity-pH profiles under different atmospheres also support the idea that more than one proton pathway appears to be involved in catalysis, and specific pathway(s) may be used by individual substrates.<br>Ph. D.
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Mueller, Martin J. "Leukotriene A₄ hydrolase : identification of amino acid residues involved in catalyses and substrate-mediated inactivation /." Stockholm, 2001. http://diss.kib.ki.se/2001/91-628-4934-4/.
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Tolliver, Benjamin M., Devaiah P. Shivakumar, and Cecelia A. McIntosh. "Effects of Amino Acid Insertion on the Substrate and Regiospecificity of a Citrus paradisi Glucosyltransferase." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etsu-works/345.
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Glucosyltransferases, or GTs, are enzymes which perform glucosylation reactions. These glucosylation reactions involve attaching a UDP-activated glucose molecule to acceptor molecules specific to the enzyme. The products of these reactions are observed to have a myriad of effects on metabolic processes, including stabilization of structures, solubility modification, and regulation of compound bioavailability. The enzyme which our lab focuses its research on is a flavonol-specific 3-O-GT found in Citrus paradisi, or grapefruit. This enzyme is part of the class of enzymes known as flavonoid GTs, which are responsible for, among other things, the formation of compounds which can affect the taste of citrus. Our lab focuses its research on performing site-directed mutagenesis on Citrus paradisi 3-O-GT in an attempt to modify its substrate specificity and regiospecificity. In this poster, we report our findings thus far concerning the addition of specific residues to the 3-O-GT's amino acid sequence based on an alignment with the sequence of a putative flavonoid GT found in Citrus sinensis.
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Tolliver, Benjamin M., Devaiah P. Shivakumar, and Cecelia A. McIntosh. "Effects of Amino Acid Sequence Insertion on the Substrate Preference of a Citrus Paradisi Glucosyltransferase." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/347.
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Glucosyltransferases (GTs) are enzymes which perform glucosylation reactions, which involve attaching a UDP-activated glucose molecule to acceptor molecules specifi c to the enzyme. The enzyme which our lab focuses its research on is a fl avonol-specifi c 3-OGT found in Citrus paradisi, or grapefruit (Cp3GT). This enzyme is part of the class of enzymes known as fl avonoid GTs, which are responsible for, among other things, the formation of compounds which can affect the taste of citrus. Our lab focuses its research on performing site-directed mutagenesis on Cp3GT in an attempt to discover the residues important for substrate and regiospecifi city. In this study, we are testing the basis of substrate septicity of Cp3GT. We hypothesize that incorporation of fi ve amino acids specifi c to Citrus sinensis GT (CsGT) into Cp3GT at 308th position may facilitate mCp3GT to use anthocyanidins as one of the substrates. We report our fi ndings thus far concerning the addition of specifi c residues to the Cp3GT’s amino acid sequence based on an alignment with the sequence of a putative fl avonoid GT found in Citrus sinensis.
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Black, Donald Lee. "Modulation of the calcium binding properties of calmodulin via amino acid replacement and target interaction /." The Ohio State University, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=osu1486397841221454.
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Pook, P. C. K. "Ligand binding and electrophysiological studies of excitatory amino acid receptors in the rat central nervous system." Thesis, University of Bristol, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381675.
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Belk, Jonathan Philip. "A Characterization of Substrates and Factors Involved in Yeast Nonsense-Mediated mRNA Decay: A Dissertation." eScholarship@UMMS, 2002. https://escholarship.umassmed.edu/gsbs_diss/65.
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Many intricate and highly conserved mechanisms have evolved to safeguard organisms against errors in gene expression. The nonsense-mediated mRNA decay pathway (NMD) exemplifies one such mechanism, specifically by eliminating mRNAs containing premature translation termination codons within their protein coding regions, thereby limiting the synthesis of potentially deleterious truncated polypeptides. Studies in Saccharomyces Cerevisiae have found that the activity of at least three trans-acting factors, known as UPF1, UPF2/NMD2, and UPF3is necessary for the proper function of the NMD pathway. Further research conducted in yeast indicates that the degradation of substrates of the NMD pathway is dependent on their translation, and that the sub-cellular site of their degradation in the cytoplasm.
Although most evidence in yeast suggests that substrates of the NMD pathway are degraded in the cytoplasm while in association with the translation apparatus, some mammalian studies have found several mRNAs whose decay appears to occur within the nucleus or before their transport to the cytoplasm has been completed. In addition, study of the mammalian TPI mRNA found that this transcript was unavailable as a substrate for the NMD pathway once it had been successfully exported to the cytoplasm, further supporting the notion that the degradation of mammalian substrates of the NMD pathway occurs in association with the nucleus, or during export from the nucleus to the cytoplasm.
To determine if yeast cytoplasmic nonsense-containing mRNA can become immune to the NMD pathway we examined the decay kinetics of two NMDS substrate mRNAs in response to repressing or activating the NMD pathway. Both the ade2-1 and pgk1-UAG-2nonsense-containing mRNAs were stabilized by repressing this pathway, while activation of NMD resulted in the rapid and immediate degradation of each transcripts. These findings demonstrate that nonsense-containing mRNAs residing in the nucleus are potentially susceptible to NMD at each round of translation.
The remainder of this thesis utilizes protein overexpression studies to gain understanding into the function of factors related to the processes of nonsense-mediated mRNA decay and translation in Saccharomyces cerevisiae. Overexpression of a C-terminal truncated form of Nmd3p was found to be dominant-negative for cell viability, translation and the normal course of rRNA biogenesis.
Overexpression studies conducted with mutant forms of the nonsense-mediated mRNA decay protein Upf1p, found that overexpression of mutants in the ATP binding and ATP hydrolysis region ofUpflp were dominant-negative for growth in an otherwise wild-type yeast strain. Furthermore, overexpression of the ATP hydrolysis mutant of Upf1p (DE572AA), resulted in the partial inhibition of NMD and a general perturbation of the translation apparatus. These results support previous studies suggesting a general role for Upf1p function in translation.
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Garza, John Anthony. "Structural and ligand-binding properties of a dual substrate specific enzymes from schizosaccharomyces pombe a dissertation /." San Antonio : UTHSC, 2009. http://learningobjects.library.uthscsa.edu/cdm4/item_viewer.php?CISOROOT=/theses&CISOPTR=45&CISOBOX=1&REC=17.
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Barcelona, Stephanie Suazo. "Investigation of the Mechanism of Substrate Transport by the Glutamate Transporter EAAC1." Scholarly Repository, 2007. http://scholarlyrepository.miami.edu/oa_theses/91.
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The activity of glutamate transporters is essential for the temporal and spatial regulation of the neurotransmitter concentration in the synaptic cleft which is critical for proper neuronal signaling. Because of their role in controlling extracellular glutamate concentrations, dysfunctional glutamate transporters have been implicated in several neurodegenerative diseases and psychiatric disorders. Therefore, investigating the mechanism of substrate transport by these transporters is essential in understanding their behavior when they malfunction. A bacterial glutamate transporter homologue has been successfully crystallized revealing the molecular architecture of glutamate transporters. However, many important questions remain unanswered. In this thesis, I will address the role of D439 in the binding of Na+, and I will identify other electrogenic steps that contribute to the total electrogenicity of the transporter cycle. The role of D439 in the binding of Na+ to the transporter was explored previously in this lab. While it was proposed that the effect of D439 in Na+ binding is indirect, the results described in this thesis provides added support to this work. Here, I will show that the D439 mutation changed the pharmacology of EAAC1 such that THA was converted from a transported substrate to a competitive inhibitor. I will also show that Na+ binding to the substrate-bound mutant transporter occurred with the same affinity as that of Na+ to the substrate-bound wild-type transporter. Therefore, based on these results, D439 is not directly involved in the binding of Na+ to the substrate-bound transporter, but that its effect is rather indirect through changing the substrate binding properties. Na+ binding steps to the empty transporter and to the glutamate-bound EAAC1 contribute only 20% of the total electrogenicity of the glutamate transporter reactions cycle. While K+-induced relocation has been proposed to be electrogenic, there is no experimental evidence that supports it. In this work, I will show that the K+-induced relocation of the empty transporter is electrogenic. Moreover, the results in this work show that the K+-dependent steps are slower than the steps associated with the Na+/glutamate translocation suggesting that the K+-induced relocation determines the transporter?s properties at steady state.
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Henderson, Jeremy. "Investigation of Saccharomyces cerevisiae Trm10 tRNA methyltransferase (m1G9) activity substrate specificity and essential amino acid residues for catalysis /." Connect to resource, 2009. http://hdl.handle.net/1811/37269.
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Sathanantham, Preethi, Shiva K. Devaiah, and Cecelia A. McIntosh. "Structure-Function Analysis of Grapefruit Glucosyltransferase Protein – Identification of Key Amino Acid Residues for its Rigid Substrate Specificity." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/352.
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Flavonoids are an important class of secondary metabolites widely distributed in plants. The majority of naturally occurring flavonoids are found in glucosylated form. Glucosyltransferases are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor flavonoid substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi (Cp3OGT) has strict substrate and regiospecificity. In this study, amino acid residues that could potentially alter the rigidity observed in this enzyme were mutated to position equivalent residues of a putative anthocyanin specific glucosyltransferase from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling followed by site directed mutagenesis to identify candidate regions, three double mutations were made. To test the basis of substrate specificity, biochemical analysis of the three recombinant mutant proteins was carried out. Recombinant protein with mutation S20G+T21S revealed that the enzyme retained activity similar to the wildtype (Cp3OGT) (WT- Km app-104.8 µM; Vmax = 24.6 pmol/min/µg, Mutant- Km app-136.42 µM; Vmax -25pmol/min/µg) but the mutant was more thermostable compared to the WT. The (S290C+S319A) mutant protein retained 40% activity relative to wildtype and has an optimum pH shifted towards the acidic side (pH 6) (Km app-8.27 µM; Vmax-90.9 pmol/min/µg). Mutation of Glutamine87 and Histine154 (H154Y+Q87I) have rendered this recombinant protein inactive with every class of flavonoid tested. Interestingly, the single point mutations H154Y and Q871I had significant activity, slightly greater than that of wildtype enzyme. The two active recombinant proteins will further be analyzed to determine whether the mutations have altered regiospecificity of the original enzyme. Product identification is being conducted using HPLC.
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Shafqat, Naeem. "Substrate specificities and functional properties of human short-chain dehydrogenases/reductases /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-829-7.
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Nygren, Patrik. "De Novo Design and Characterization of Surface Binding Peptides - Steps toward Functional Surfaces." Licentiate thesis, Linköping University, Linköping University, Sensor Science and Molecular Physics, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-8992.
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<p>The ability to create surfaces with well-defined chemical properties is a major research field. One possibility to do this is to design peptides that bind with a specific secondary structure to silica nanoparticles. The peptides discussed in this thesis are constructed to be random coil in solution, but are “forced” to become helical when adsorbed to the particles. The positively charged side-chains on the peptides strongly disfavor an ordered structure in solution due to electrostatic repulsion. When the peptides are introduced to the particles these charges will strongly favor the structure because of ion pair bonding between the peptide and the negatively charged nanoparticles. The peptide-nanoparticle system has been thoroughly investigated by systematic variations of the side-chains. In order to determine which factors that contributes to the induced structure, several peptides with different amino acid sequences have been synthesized. Factors that have been investigated include 1) the positive charge density, 2) distribution of positive charges, 3) negative charge density, 4) increasing hydrophobicity, 5) peptide length, and 6) by incorporating amino acids with different helix propensities. Moreover, pH dependence and the effect of different nanoparticle curvature have also been investigated. It will also be shown that the system can be modified to incorporate a catalytic site that is only active when the helix is formed. This research will increase our understanding of peptide-surface interactions and might be of importance for both nanotechnology and medicine.</p>
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Chelliah, Vijayalakshmi. "Functional restraints on amino acid substitution patterns : application to definition of binding sites and sequence-structure homology recognition." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615170.
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Niemand, Jandeli. "A phage display study of interacting peptide binding partners of malarial S-Adenosylmethionine decarboxylase/Ornithine decarboxylase." Diss., University of Pretoria, 2007. http://hdl.handle.net/2263/24105.
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Due to the increasing resistance against the currently used antimalarial drugs, novel chemotherapeutic agents that target new metabolic pathways for the treatment of malarial infections are urgently needed. One approach to the drug discovery process is to use interaction analysis to find proteins that are involved in a specific metabolic pathway that has been identified as a drug target. Protein-protein interactions in such a pathway can be preferential targets since a) there is often greater structural variability in protein-protein interfaces, which can lead to more effective differentiation between the parasite and host proteins; and b) the important amino acids in a protein-protein interface are often conserved and even one amino acid mutation can lead to the dissociation of the complex, implying that resistance should be slower to appear. Since polyamines and their biosynthetic enzymes occur in increased concentrations in rapidly proliferating cells, the inhibition of polyamine metabolism is a rational approach for the development of antiparasitic drugs. Polyamine synthesis in P. falciparum is uniquely facilitated by a single open reading frame that encodes both rate-limiting enzymes in the pathway, namely ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). The AdoMetDC/ODC domains are assembled in a heterotetrameric bifunctional protein complex of ~330 kDa. Inhibition of both decarboxylase activities is curative of murine malaria and indicates the viability of such strategies in malaria control. It was hypothesized that protein ligands to this enzyme can be utilized in targeting the polyamine biosynthetic pathway in a novel approach. The bifunctional PfAdoMetDC/ODC was recombinantly expressed with a C-terminal Strep-tag-II to allow affinity purification. Subsequent gel electrophoresis analysis showed the presence of 3 contaminating proteins (~60 kDa, ~70 kDa and ~112 kDa) that co-elute with the ~330 kDa AdoMetDC/ODC. Efforts to purify the bifunctional protein to homogeneity included subcloning into a double-tagged vector for tandem affinity purification as well as size-exclusion HPLC. SDS-PAGE analysis of these indicated that separation of the four proteins was not successful, implicating the presence of strong protein-protein interactions. Western blot analysis showed that the ~112 kDa and ~70 kDa peptides were recombinantly produced with a C-terminal Strep-tag, indicating their heterologous origin. The ~60 kDa fragment was however not recognised by the tag-specific antibodies. This implies that this fragment is of E. coli origin. MS-analysis of the contaminating bands showed that the ~112 kDa peptide is an N-terminally truncated form of the full-length protein, the ~70 kDa peptide is a mixture of N-terminally truncated recombinant protein and E. coli DnaK and the ~60 kDa peptide is E. coli GroEL. A P. falciparum cDNA phage display library was used to identify peptide ligands to PfAdoMetDC/ODC. Of the peptides isolated through the biopanning process, only one was shown to occur in vivo. It could however not be conclusively shown that the isolated peptides bind to PfAdoMetDC/ODC and not to the co-eluting E. coli proteins. It is thought that while it is extremely likely that interacting protein partners to PfAdoMetDC/DOC exist, the available technologies are not sufficient to lead to the identification of such partners.<br>Dissertation (MSc (Biochemistry))--University of Pretoria, 2008.<br>Biochemistry<br>unrestricted
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Tran, David. "Investigating the substrate specificity of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase." Thesis, University of Canterbury. Chemistry, 2011. http://hdl.handle.net/10092/6565.
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The shikimate pathway is a biosynthetic pathway that is responsible for producing a variety of organic compounds that are necessary for life in plants and microorganisms. The pathway consists of seven enzyme catalysed reactions beginning with the condensation reaction between D-erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP) to give the seven-carbon sugar DAH7P. This thesis describes the design, synthesis and evaluation of a range of alternative non-natural four-carbon analogues of E4P (2- and 3-deoxyE4P, 3-methylE4P, phosphonate analogues of E4P) to probe the substrate specificity of different types of DAH7P synthases [such as Mycobacterium tuberculosis (a type II DAH7PS), Escherichia coli (a type Ialpha DAH7PS) and Pyrococcus furiosus (a type Ibeta DAH7PS)].
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St-Jacques, Antony D. "Engineering of Multi-Substrate Enzyme Specificity and Conformational Equilibrium Using Multistate Computational Protein Design." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/38590.
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The creation of enzymes displaying desired substrate specificity is an important objective of enzyme engineering. To help achieve this goal, computational protein design (CPD) can be used to identify sequences that can fulfill interactions required to productively bind a desired substrate. Standard CPD protocols find optimal sequences in the context of a single state, for example an enzyme structure with a single substrate bound at its active site. However, many enzymes catalyze reactions requiring them to bind multiple substrates during successive steps of the catalytic cycle. The design of multi-substrate enzyme specificity requires the ability to evaluate sequences in the context of multiple substrate-bound states because mutations designed to enhance activity for one substrate may be detrimental to the binding of a second substrate. Additionally, many enzymes undergo conformational changes throughout their catalytic cycle and the equilibrium between these conformations can have an impact on their substrate specificity. In this thesis, I present the development and implementation of two multistate computational protein design methodologies for the redesign of multi-substrate enzyme specificity and the modulation of enzyme conformational equilibrium. Overall, our approaches open the door to the design of multi-substrate enzymes displaying tailored specificity for any biocatalytic application.
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Cherry, Melissa A. "Sequence dependence of the activity of amphipathic peptides." View electronic thesis, 2008. http://dl.uncw.edu/etd/2008-2/rp/cherrym/melissacherry.pdf.
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Trott, Amy Elizabeth. "Amino Acid Residues in LuxR Critical for its Mechanism of Transcriptional Activation during Quorum Sensing." Thesis, Virginia Tech, 2000. http://hdl.handle.net/10919/34070.
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<I>Vibrio fischeri</I>, a symbiotic bioluminescent bacterium, serves as one of the best understood model systems for a mechanism of cell-density dependent bacterial gene regulation known as quorum sensing. During quorum sensing in <I>V. fischeri</I>, an acylated homoserine chemical signal (autoinducer) is synthesized by the bacteria and used to sense their own species in a given environment. As the autoinducer levels rise, complexes form between the autoinducer and the N-terminal domain of a regulatory protein, LuxR. In response to autoinducer binding, LuxR is believed to undergo a conformational change that allows the C-terminal domain to activate transcription of the luminescence or <I>lux</I> operon. To further understand the mechanism of LuxR-dependent transcriptional activation of the <I>lux</I> operon, PCR-based site-directed mutagenesis procedures have been used to generate alanine-substitution mutants in the C-terminal forty-one amino acid residues of LuxR, a region that has been hypothesized to play a critical role in the activation process. An <I>in vivo</I> luminescence assay was first used to test the effects of the mutations on LuxR-dependent activation of the <I>lux</I> operon in recombinant <I>Escherichia coli</I>. Luciferase levels present in cell extracts obtained from these strains were also quantified and found to correlate with the luminescence results. Eight strains encoding altered forms of LuxR exhibited a "dark" phenotype with luminescence output less than 50% and luciferase levels less than 50% of the wildtype control strain. Western immunoblotting analysis with cell extracts from the luminescence and luciferase assays verified that the altered forms of LuxR were expressed at levels approximately equal to wildtype. Therefor, Low luminescence and luciferase levels could be the result of a mutation that either affects the ability of LuxR to recognize and bind its DNA target (the <I>lux</I> box) or to establish associations with RNA polymerase (RNAP) at the <I>lux</I> operon promoter necessary for transcriptional initiation. A third <I>in vivo </I>assay was used to test the ability of the altered forms of LuxR to bind to the <I>lux</I> box (DNA binding assay/repression). All of the LuxR variants exhibiting the "dark" phenotype in the luminescence and luciferase assay were also found to be unable to bind to the <I>lux</I> box in the<I> </I>DNA binding assay. Therefore, it can be concluded that the alanine substitutions made at these positions affect the ability of LuxR to bind to the <I>lux</I> box in the presence and absence of RNA polymerase. Another class of mutants exhibited wildtype phenotypes in the luminescence and luciferase assays but were unable to bind to the <I>lux</I> box in the DNA binding assay. The alanine substitutions made at these amino acid residues may be making contacts with RNAP that are important for maintaining the stability of the DNA binding region of LuxR. Alanine substitutions made at these positions have a defect in DNA binding at the promoter of the <I>lux</I> operon only in the absence of RNAP. None of the alanine substitutions made in the C-terminal forty-one amino acids of LuxR were found to affect activation of transcription of the <I>lux</I> operon without also affecting DNA binding. Taken together, these results support the conclusion that the C-terminal forty-one amino acids of LuxR are important for DNA recognition and binding of the <I>lux</I> box rather than positive control of the process of transcription initiation.<br>Master of Science
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Adepoju, Olusegun A., Devaiah K. Shiva, and Cecelia A. McIntosh. "Using Site-Directed Mutagenesis to Determine Impact of Amino Acid Substitution on Substrate and Regiospecificity of Grapefruit Flavonol 3-O-Glucosyltransferase." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etsu-works/346.
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Flavonoids are secondary metabolites that are important in plant defense, protection and human health. Most naturally-occurring flavonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. A flavonol-specific 3-O-GT enzyme has been identified and cloned from leaf tissues of grapefruit. The enzyme shows rigid substrate specificity and regiospecificity. F3-O-GTs from grape (Vitis vinifera) and grapefruit (Citrus paradisi) were modeled against F7-O-GTs from Crocus sativus and Scrutellaria biacalensis, and several non-conservative amino acid differences were identified that may impact regioselectivity. This research is designed to test the hypothesis that specific amino acid residues impart the regiospecificity of the grapefruit enzyme. Site-directed mutagenesis was performed on three potentially key amino acid residues within the grapefruit F3-O-GT that were identified through homology modeling. Analyses of the enzyme activity of the mutant F3-O-GT proteins revealed that the single point mutations of serine 20 to leucine (S20L) and proline 297 to phenylalanine (P297F) rendered the recombinant enzyme inactive with flavonol substrates. Mutation of glycine 392 to glutamate (G392E) was active at 80% relative to the wild type. The mutant enzyme also did not show broadened acceptor specificity as it also favored flavonols as the preferred acceptor substrate. The glucosylation products of the active mutant enzyme will be analyzed to determine if this resulted in a change in regiospecificity.
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Williams, Kyle Brandon. "The DamX cell division protein of Escherichia coli: identification of amino acid residues critical for septal localization and peptidoglycan binding." Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/625.
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In the bacterium Escherichia coli, cell division involves the concerted inward growth of all three layers of the cell envelope: the cytoplasmic membrane, the peptidoglycan (PG) cell wall, and the outer membrane. This is a complex, highly regulated process that involves over 20 proteins. Four of these proteins contain a domain of ~70 amino acids known as a SPOR domain (Pfam no. 05036). One of these SPOR domains (from a protein named FtsN) has been shown previously to bind PG. In this thesis we show that six additional SPOR domains, three from E. coli and three from other bacterial species, also bind PG. Thus, PG binding is a general activity of SPOR domains. We then examine the SPOR domain from DamX of E. coli in detail. In collaboration with Dr. Andrew Fowler of the NMR Core Facility, we determined the solution structure of the domain. The domain adopts an "RNP fold," characterized by a four-stranded anti-parallel β-sheet that is buttressed on one side by α-helixes. Several mutant forms of the DamX SPOR domain were constructed and studied both in vivo and in vitro. These studies support the following inferences: 1) The β-sheet is the PG-binding site; 2) The β-sheet contains critical information for targeting the SPOR domain to the midcell; 3) The SPOR domain probably localizes to the midcell by binding preferentially to septal PG; and 4) It follows, then, that septal PG must differ from PG elsewhere around the cell. We suggest that further studies of the SPOR:PG interaction will yield novel insights into PG biogenesis during septation.
This thesis also presents an in vivo characterization of several mutant forms of a cytoplasmic membrane protein named FtsW, homologs of which are found in all bacteria that contain a PG cell wall. FtsW recruits a PG synthase named FtsI to the division site and might also transport PG precursors across the cytoplasmic membrane. We systematically mutagenized each of FtsW's ten transmembrane (TM) helixes and investigated the ability of the mutant proteins to support division, localize to the division site, and recruit FtsI. This characterization leads us to propose that TM1 is involved in targeting FtsW to the division site, TM4 is involved in the putative transport activity, and TM10 is involved in recruitment of FtsI.
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Emami, Fatemesadat. "Thermodynamically Consistent Interatomic Potentials for Silica to Design Specifically Binding Peptides: Role of Surface Chemistry, PH, and Amino Acid Sequence." University of Akron / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=akron1366597654.
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Kobayashi, Takuya. "Amino acid residues conferring ligand binding properties of prostaglandin I and prostaglandin D receptorsに関する研究". Kyoto University, 2000. http://hdl.handle.net/2433/151431.
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Zhang, Deqiang Roberts Richard W. Goddard William A. "Structure-based design of mutant proteins : I. Molecular docking studies of amino acid binding to wild-type aminoacyl-tRNA synthetases. II. Structure-based design of mutant aminoacyl-tRNA synthetases for non-natural amino acid incorporation /." Diss., Pasadena, Calif. : California Institute of Technology, 2003. http://resolver.caltech.edu/CaltechETD:etd-12182002-190040.
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Papp, Laura V., and n/a. "Multiple Levels of Regulation of Human SECIS Binding Protein 2, SBP2." Griffith University. School of Biomolecular and Biomedical Science, 2006. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20070208.145623.
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Selenium is an essential trace mineral of fundamental importance to human health. Its beneficial functions are largely attributed to its presence within a group of proteins named selenoproteins in the form of the amino acid selenocysteine (Sec). Recently, it was revealed that the human selenoproteome consists of 25 selenoproteins, and for many of them their function remains unknown. The most prominent known roles of selenoproteins are to maintain the intracellular redox homeostasis, redox regulation of intracellular signalling and thyroid hormone metabolism. Sec incorporation into selenoproteins employs a unique mechanism that involves decoding of the UGA stop codon. The process requires interplay between distinct, intrinsic features such as the Sec Insertion Sequence (SECIS) element, the tRNASec and multiple protein factors. The work presented in this thesis has focused on characterising the regulation of human SECIS binding protein 2, SBP2, a factor central to this process. Experimental approaches combined with bioinformatics analysis revealed that SBP2 is subjected to alternative splicing. A total of nine alternatively spliced transcripts appear to be expressed in cells, potentially encoding five different protein isoforms. The alternative splicing events are restricted to the 5?-region, which is proposed to be dispensable for Sec incorporation. One of the variants identified, contains a mitochondrial targeting sequence that was capable of targetting SBP2 into the mitochondrial compartment. This isoform also appears to be expressed endogenously within the mitochondria in cells. Previous reports have depicted SBP2 as a ribosomal protein, despite the presence of a putative Nuclear Localisation Signal (NLS). In this study it was found that SBP2 subcellular localisation is not restricted to ribosomes. Intrinsic functional NLS and Nuclear Export Signals (NESs), enable SBP2 to shuttle between the nucleus and the cytoplasm via the CRM1 pathway. In addition, the subcellular localisation of SBP2 appears to play an important role in regulating Sec incorporation into selenoproteins. The subcellular localisation of SBP2 is altered by conditions imposing oxidative stress. Several oxidising agents induce the nuclear accumulation of SBP2, which occurs via oxidation of cysteine residues within a novel redox-sensitive cysteine rich domain (CRD). Cysteine residues were to form disulfide bonds and glutathione-mixed disulfides during oxidising conditions, which are efficiently reversed in vitro by the thioredoxin and glutaredoxin systems, respectively. These modifications negatively regulate selenoprotein synthesis. Cells depleted of SBP2 are more sensitive to oxidative stress than control cells, which correlated with a substantial decrease in selenoprotein synthesis after treatment with oxidising agents. These results provide direct evidence that SBP2 is required for Sec incorporation in vivo and suggest that nuclear sequestration of SBP2 under such conditions may represent a mechanism to regulate the expression of selenoproteins. Collectively, these results suggest that SBP2 is regulated at multiple levels: by alternative splicing, changes in subcellar localisation and redox control.
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Adepoju, Olusegun Adeboye. "Using Site-Directed Mutagenesis to Determine Impact of Amino Acid Substitution on Substrate and Regiospecificity of Grapefruit Flavonol Specific 3-O-Glucosyltransferase." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etd/2404.
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Flavonoids are secondary metabolites that are important in plant defense, protection, and human health. Most naturally-occurring flavonoids are found in glucosylated forms. Glucosyltransferases catalyze the transfer of glucose from high-energy sugar donors to an acceptor molecule. The grapefruit flavonol-specific 3-O-glucosyltransferase (F3-O-GT) is highly substrate and regio-specific. The goal of this research is to unravel the amino acid residues responsible for the grapefruit enzyme’s rigid specificity, while attempting to alter the regiospecific glucosylation pattern through site-directed mutagenesis and homology modeling. This research tested the hypothesis that substitution of potential key amino acid residues within the grapefruit Cp-F3-O-GT with position equivalent residues within F7-O-GTs would alter the 3-O-glucosylation of the enzyme. Results reveal that specific single point mutations of residues are capable of abolishing enzymatic activity. Recombinant mutant G392E retained activity and showed an increased affinity for kaempferol relative to the wild-type; however, the rigid regiospecific glucosylation pattern of the enzyme was retained.
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Smith, Robert Peter. "Exploration of the active site of Staphylococcus aureus neuraminic acid lyase using non-canonical amino acids and their effects on substrate specificity." Thesis, University of Leeds, 2017. http://etheses.whiterose.ac.uk/20719/.
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The cost of chemical synthesis of pharmaceuticals contributes significantly to their final price and part of this cost is incurred due to use of extreme temperatures and pressures required by some traditional catalysts. The interest in catalysis using enzymes, or biocatalysis, from industry has been growing recently, due to enzymes’ ability to work at room temperature and pressure, and the reduction in toxic solvent waste produced from an enzyme reaction compared to a traditionally catalysed reaction. The specificity of enzymes, while useful in product formation, can make applying them to synthetic chemistry challenging due to the restriction this causes in substrates that each enzyme accepts. This can often be avoided by amino acid mutagenesis, but when this is performed genetically, only 20 different amino acids can be used. Non-canonical amino acids (ncAAs) have the potential to enhance properties of enzymes, such as enzyme stability and substrate specificities, to hitherto unseen extremes, due to the massive diversity of amino acids outside the canonical 20. 900 enzyme-aldehyde pairs were screened for activity, and Y252Lanthionine was found to catalyse the aldol reaction between pyruvate and glucuronolactone better than the wild type enzyme for the same reaction. Upon crystallisation, this enzyme was found to be a mixture of both L- and D-stereoisomers at the protein backbone where the ncAA was inserted. Computational experiments were performed to assess the substrate binding capability of the modified enzyme and the wild-type enzyme. The modified side chain holds the substrate more tightly than the wild-type side chain, contributing to increased residence time in the active site.
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Gilbert, Michele. "Design of synthetic peptides that display cell binding and signaling sequences on calcium phosphate surfaces /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/8063.
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Nordling, Erik. "Biocomputational studies on protein structures /." Stockholm, 2002.
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Bennett, Allison E. "Characterization of sortase and its effect on the virulence of Streptococcus pneumoniae." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2008d/bennet.pdf.
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Vichitphan, Kanit. "Azotobacter vinelandii Nitrogenase: Effect of Amino-Acid Substitutions at the Alpha Gln-191 Residue of the MoFe Protein on Substrate Reduction and CO Inhibition." Diss., Virginia Tech, 2001. http://hdl.handle.net/10919/11266.
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The FeMo cofactor is one of two types of prosthetic group found in the larger of the two nitrogenase component proteins, called the MoFe protein, and it is strongly implicated as the substrate binding and reduction site. The glutamine-191 residue in the Alpha-subunit of the MoFe protein of A. vinelandii nitrogenase was targeted for substitution because its side chain is involved in a hydrogen-bond network from one of the terminal carboxylates of the homocitrate component of FeMo cofactor through to the backbone NH of Alpha Gly-61, which is adjacent to Alpha Cys-62, which ligates to the P cluster (the second type of prosthetic group in the MoFe protein).
A variety of altered MoFe proteins produced by the A. vinelandii mutant strains, namely the Alpha Pro-191, Alpha Ser-191, Alpha Thr-191, Alpha His-191, Alpha Glu-191, and Alpha Arg-191 altered MoFe proteins, have been purified to homogeneity and the catalytic properties of these altered MoFe proteins have been compared to those of wild type MoFe protein. Unlike wild type, the six altered MoFe proteins have decreased catalytic activity on substrate reduction and exhibited H2 evolution that was partially inhibited by added CO. Moreover, some of altered MoFe proteins with lower specific activity for the C2H4 production can produce C2H6 from C2H2.
The results from the pH and activity studies indicate that the substitutions on the MoFe protein have an effect on the contribution of the responsible acid-base group(s) involved in proton transfer for H+- and C2H2-reduction. Furthermore, the inhibition by CO of hydrogen evolution by these altered MoFe proteins is likely from a lowering of the rate of both electron and proton transfer to the H+- reduction site(s).
Some altered MoFe proteins but not wild type MoFe protein can produce C2H6 from C2H2. This observation suggested a lower apparent binding affinity for C2H2 and a slower proton transfer to C2H2 reduction with these altered MoFe proteins, which allow the intermediate to stay at the site longer and be further reduced by two electrons and two protons to give C2H6.
These changes in the biochemical properties of these altered MoFe proteins indicate that the Alpha Gln-191 residue is intimately involved in substrate binding and reduction including proton delivery to substrate.<br>Ph. D.
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Pedersen, Sindre Andre. "Metal binding proteins and antifreeze proteins in the beetle Tenebrio molitor : a study on possible competition for the semi-essential amino acid cysteine." Doctoral thesis, Norwegian University of Science and Technology, Department of Biology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-1504.
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<p>In their natural environment animals are confronted by both physical (eg. extreme temperatures, desiccation) and chemical stressors (e.g. pollutants). Stress may be defined as a condition that is evoked in an organism by one or more environmental factors that bring the organism near to or over the edges of its ecological niche (van Straalen 2003). Various defence systems exist to cope with different forms of stress and restore homeostasis. Often, production of various proteins or enzymes are involved in these defence systems (Korsloot et al. 2004). Since an organism’s resources may be considered to be limited, the ability to restore homeostasis depends on the severity of the different forms of stress it experiences. It has been proposed that pollutants present in the environment may alter the ability to respond to climatic stressors like e.g. low temperature, desiccation (Holmstrup 2002).</p><p>This work deals with the possible consequences of combined stress from metal exposure and low temperature in cold hardy insects. Many of these insects produce so called <i>antifreeze proteins</i> that protect them from lethal freezing. Metallothioneins are metal binding proteins that are considered to be important in detoxification when animals are exposed to metals. Metallothioneins and most forms of antifreeze proteins from insects are known to contain unusually high amounts cysteine. Cysteine is considered to be semi-essential, since it must be derived from the essential amino acid methionine (Choen 2004). Induction of one of these two types of proteins may potentially deplete the cysteine pool and thus reduce the capacity to produce the other type. Alternatively, the animals might have evolved other structures to avoid a potential competition for cysteine. The purpose of the present work was to explore these possible scenarios.</p><br>Paper I and II reprinted with kind permission of Elsevier, Sciencedirect.com
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Keyari, Charles Mambo. "Glycosylating Enkephalins: Design, Glycosylation Using Sugar Acetates in the Preparation of Glycosyl Amino Acids for Glycopeptide Syntheses, Binding at the Opioid Receptors and Analgesic Effects." Diss., The University of Arizona, 2007. http://hdl.handle.net/10150/193652.
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Improved procedures for the glycosylation of serine and threonine utilizing Schiff base activation are reported. The procedures are less expensive and more efficient alternatives to previously published methods. The Schiff bases exhibited ring-chain tautomerism in CDCl₃ as shown by ¹H NMR. Acting as glycosyl acceptors, the Schiff bases reacted at RT with simple sugar peracetate donors with BF₃•OEt₂ promotion to provide the corresponding protected amino acid glycosides in good yields. With microwave irradiation, the reactions were complete in 2-5 minutes. Glycosylation with the dipeptide Schiff base shows the potential of this method in the preparation of peptide building blocks. To investigate this reaction further, direct glycosylation of sugar acetates with FMOC-Ser-OH/OBZl under BF₃•OEt₂ promotion in a microwave provided glycosides in high yield. In addition to the expected glycoside products acetylated side products resulting from acetate migration were isolated, suggesting that activation of the anomeric sugar acetates with a Lewis acid such BF₃•OEt₂ led to an oxocarbenium ion, which rearranged to a 1,2-dioxocarbenium ion because of the acetate participating group at C-2. Solvent participation was also illustrated with acetate migration being more pronounced when CH₃CN was used as a solvent and resulted in less product yield and higher amounts of the acetylated product. The acyl transfer products in these reactions where sugar acetates serve as glycosyl donors is reported for the first time, which also implies that ortho-ester like intermediates are important in the reaction mechanism. Keeping the message segment constant in the sequence H-Tyr-DThr-Gly-Phe-Leu- Ser-CO-NH₂ and modification of the address segment with different carbohydrate moieties had little effect on selectivity for binding at the μ, δ, or κ-opiod receptors. However, substitution of D-threonine with D-serine or the less polar D-alanine in the message segment resulted in a loss of κ-receptor affinity. Further replacement of D-threonine with the more hydrophobic D-valine resulted in complete loss of κ-binding affinity generating pure μ-δ agonists. These data suggests that changes in the message segment of the pharmacophore results in the glycopeptide adopting a conformation that is less favorable for -binding receptor activity. Finally, the peripheral administration and i.c.v. tests of the drugs suggest that modifications in the message segment of the pharmacophore influences the potency of these compounds.
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Wang, Haihong. "Amino acid residues constituting the agonist binding site of the human P2X3 receptor and subunit stoichiometry of heteromeric P2X2/3 and P2X2/6 receptors." Doctoral thesis, Universitätsbibliothek Leipzig, 2013. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-112913.
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Homotrimeric P2X3 and heteromeric P2X2/3 receptors are present in sensory ganglia and participate in pain perception. In order to develop pharmacological antagonists for these receptors, it is important to clarify which amino acid (AA) residues constitute the agonist binding pouch as well as to learn the stoichiometry of the receptor subunits forming a heteromeric receptor. We expressed the homomeric human (h)P2X3 receptor or its mutants in HEK293 cells and measured the ATP-induced responses by the whole-cell patch-clamp method. For the binding-site mutants, all conserved and some non-conserved AAs in the four nucleotide binding segments (NBSs) of the P2X3 subunit were sequentially replaced by alanine. Especially the positively charged AAs Lys and Arg appeared to be of critical importance for the agonist effects. We concluded that groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the P2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor. We were also interested to find out, whether two heteromeric receptors (P2X2/3 and P2X2/6), where P2X2 combines with two different partners, have an obligatory subunit stoichiometry of 1:2 or whether the subunit stoichiometry may be variable. For this purpose we used non-functional P2X2, P2X3 and P2X6 subunit-mutants to investigate the composition of heteromeric P2X2/3 and P2X2/6 receptors. The subunit stoichiometry of P2X2/3 and P2X2/6 was found to be 1:2 and 2:1, respectively. Thus, recognitions sites between P2X2 and its partners rather than random association may govern the subunit compositions of the receptor trimers.
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Ruhlman, Tracey. "Determinants of Chloroplast Gene Expression and Applications of Chloroplast Transformation in Lactuca Sativa and Nicotiana Tabacum." Doctoral diss., University of Central Florida, 2009. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2854.
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Genetic modification of plastids in the model plant tobacco (Nicotiana tabacum) has demonstrated that numerous foreign gene products can accumulate to high levels in this setting. Plastid biotechnology is maturing to encompass the improvement of food and feed species and the production of biopharmaceutical proteins for oral delivery necessitating development of stable transplastomic edible plants. In the interest of establishing an edible platform we have investigated the use of native and foreign regulatory elements in relation to foreign gene expression in plastids. Multiple sequence alignments of intergenic regions for 20 species of angiosperm showed that despite 95% identity in the coding region, identity in the psbA upstream region is 59% across all taxa examined, other gene coding regions displayed sequence identity of 80-97%, whereas the non-coding regions were 45-79% suggesting that our physical data can be extrapolated beyond the model presented. We found that by exchanging psbA untranslated regions (UTRs) between N. tabacum and lettuce (Lactuca sativa), the expression of the CTB-proinsulin (CTB-Pins) monocistronic transcript declined by 84% and foreign protein accumulation was reduced by as much as 97% in mature leaves. Polyribosome association assays suggest that ribosome-free transgenic transcripts are stabilized where the native UTR is employed. RNA EMSA revealed that binding proteins interacted with psbA 5' UTRs in a species specific manner and the half life of the L. sativa 5'UTR-CTB-Pins mRNA was reduced by 3.7 fold in N. tabacum stromal extracts. Our data indicate that the use of species-specific regulatory elements could lead to establishment of reproducible plastid transformation in desirable target species such as L. sativa. Using transplastomic L. sativa for oral delivery of bioencapsulated CTB-Pins we delayed the onset of diabetes in NOD mice when retinyl acetate supplement was provided compared to untouched mice. In this 30 week study we monitored blood glucose levels and evaluated the in vitro suppressive capacity of regulatory T cells isolated from diabetic mice. Whether delay or prevention was achieved appeared to be a function of antigen dose as high dose resulted in a nine week delay of onset while low dose reduced the incidence of diabetes by 36%. In addition we have evaluated metabolic engineering in the N. tabacum model where we generated cis-genic lines expressing nucleus-encoded methionine pathway enzymes in plastids. Transplastomic expression of Cystathionine gamma-Synthase led to a three-fold increase in enzyme activity and a doubling of methionine content in leaves without a deleterious phenotype. In exploring molecular mechanisms supporting gene expression in plastids and applying transplastomic technology to real human problems this work seeks address the potential of plastid biotechnology for improvement of commodity crops and production of biopharmaceuticals.<br>Ph.D.<br>Department of Biomolecular Science<br>Other<br>Biomedical Sciences PhD
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Li, Min. "Bayesian discovery of regulatory motifs using reversible jump Markov chain Monte Carlo /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/9529.
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Jiang, Ruisheng. "STUDIES OF THE PYRROLYSYL-TRNA SYNTHETASE." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1365512882.
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Saliba, Elie. "A NOVEL TORC1 ACTIVATION PATHWAY STIMULATED BY THE PLASMA MEMBRANE H+-ATPASE UNRAVELED FROM THE STUDY OF SUBSTRATE-INDUCED ENDOCYTOSIS OF AMINO ACID TRANSPORTERS IN YEAST SACCHAROMYCES CEREVISIAE." Doctoral thesis, Universite Libre de Bruxelles, 2017. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/262158.
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Chez les eucaryotes, le complexe kinase TORC1 (Target Of Rapamycin Complex 1) joue un rôle central dans le contrôle de la croissance cellulaire. Il intègre de nombreux signaux et agit en modulant l’état de phosphorylation de différents effecteurs, principalement des protéines impliquées dans des processus anaboliques ou cataboliques. Parmi ces signaux, on distingue notamment les acides aminés. Ces derniers agissent sur TORC1 via l’action de protéines de la famille des GTPases Rag, elles-mêmes régulées par des facteurs GEF et GAP. Des études récentes sur des cellules mammifères ont mis en évidence l’existence de senseurs d'acides aminés, capables de moduler l'activité des facteurs GEF et GAP. Chez la levure, ces senseurs restent toutefois inconnus. Chez la levure, TORC1 contrôle la fonction de plusieurs protéines y compris des transporteurs d'acides aminés de la membrane plasmique, comme la perméase générale des acides aminés, Gap1. C’est via sa branche de signalisation Tap2-PP2A/Npr1 que TORC1 contrôle l'ubiquitylation et le trafic intracellulaire de ces transporteurs, et ceci, en modulant l’activité d’adaptateurs de type α-arrestine de l'ubiquitine-ligase Rsp5.Dans ce travail, nous avons combiné des approches de génétique et de biochimie chez la levure afin d’étudier la régulation de TORC1 et son rôle dans l'endocytose en réponse au substrat de Gap1, la perméase générale des acide aminés, et de Can1, la perméase spécifique de l'arginine.Dans la première et la deuxième partie de ce travail, je décris ma contribution à l'étude qui visait à élucider le mécanisme d'ubiquitylation de Gap1 et de Can1 induite par le transport de leurs substrats. Le modèle déduit de ce travail propose que cette régulation négative n'est pas due à l'accumulation intracellulaire des acides aminés transportés, mais à un changement conformationnel des perméases, couplé à la réaction de transport et qui fait apparaitre un état ouvert vers l'intérieur, entraînant ainsi le remodelage de la queue cytoplasmique N-terminale et en conséquence l’exposition d'un site de liaison caché pour des adaptateurs de Rsp5 de type α-arrestine. Dans le cas de Can1, l'α-arrestine principale impliquée est Art1 et doit être stimulée via TORC1. Cependant, les α-arrestines Bul1/2, impliquées dans la régulation négative de Gap1, sont capables de promouvoir son ubiquitylation induite par le transport de substrat, qu'elles aient ou non été stimulées via TORC1. Nous fournissons également des preuves que d'autres perméases d'acides aminés spécifiques (Mup1, Lyp1) sont régulées par leurs propres substrats d'une manière similaire à Can1.Dans la dernière partie de ce travail, nous avons étudié le mécanisme par lequel le transport des acides aminés chez la levure stimule l'activité de TORC1 via les GTPases Rag, Gtr1 et Gtr2. En analysant en Western Blot l’état de phosphorylation de Npr1 et Sch9, deux kinases effectrices de TORC1, nous avons révélé que le signal général qui déclenche l'activation Gtr-dépendante de TORC1 est le flux de H+ couplé au transport des acides aminés généré par des symporteurs H+/acide-aminés. Dans ce contexte, nous avons identifié la pompe à H+ de la membrane plasmique, Pma1, comme étant un régulateur essentiel de l'activité de TORC1. L'activité de transport de Pma1 étant elle-même stimulée par une augmentation des protons cytosoliques, nous suggérons que Pma1 module TORC1 par un effet de signalisation.Collectivement, nos résultats fournissent de nouvelles perspectives sur le rôle central de TORC1 dans le contrôle des transporteurs de nutriments chez la levure.<br>The Target of Rapamycin Complex 1 (TORC1) plays a pivotal role in controlling cell growth in probably all eukaryotic organisms. It operates by integrating upstream signals like growth factors and nutrients to modulate by phosphorylation multiple downstream effectors, mostly proteins involved in anabolic or catabolic processes. Among the various signals that impinge on TORC1, nitrogen sources, in particular amino acids are primordial input signals modulating TORC1 activity through the conserved Rag family of GTPases. Recent studies in mammals have shed the light on the existence of various sensor systems of internal amino acids that modulate the activity of the GEF and GAP factors acting on the Rag GTPases. Yet, in yeast the amino acid sensing events acting upstream of the Rag GTPase (named Gtr1 and Gtr2) regulators remain poorly known. Yeast TORC1 controls the function of many proteins including several plasma membrane amino acid transporters, e.g. Gap1, the general amino acid permease. It does so via the Tap2-PP2A/Npr1-signaling branch that controls the ubiquitylation and intracellular trafficking of these proteins through regulation of α-arrestin-type adaptors of the ubiquitin-ligase Rsp5. In this work, we combined yeast genetics and biochemical assays to study TORC1 regulation by amino acids and to illustrate the role of TORC1 in substrate-transport mediated endocytosis of Gap1 and of the arginine specific permease, Can1. In the first and second part of this work, I describe my contribution to the study that aimed at elucidating the mechanism of substrate-transport-mediated ubiquitylation and endocytosis of Gap1 and Can1. The model deduced from this work states that this down-regulation is not due to intracellular accumulation of the transported amino acids, but to substrate-transport-induced conformational transition of the transporters to an inward-facing state, resulting in remodeling of their N-terminal cytoplasmic tail and subsequent exposure of a hidden binding site for α-arrestin-like adaptors of Rsp5. In the case of Can1, the main α-arrestin involved is Art1 and needs be stimulated via TORC1. The Bul1/2 α-arrestins involved in Gap1 down-regulation, however, are able to promote its substrate-transport-elicited ubiquitylation regardless of whether they have been stimulated via TORC1 or not. We also provide evidence that other specific amino acid permeases (Mup1, Lyp1) are regulated by their own substrates in a manner similar to Can1. In the last part of this work, we investigated how amino acid uptake by yeast cells triggers Rag/Gtr-dependent activation of TORC1. By assaying the phosphorylation on western blot of two TORC1-downstream effectors, the Npr1 and Sch9 kinases, we showed that uptake by Gap1 of ß-alanine, which cannot be used as a nitrogen source, unexpectedly stimulates TORC1 activity. Further analysis of this response allowed us to show that the general signal triggering Gtr-dependent activation of TORC1 in response to amino acid uptake is the influx of H+ coupled to transport via H+/amino-acid symporters. Furthermore, we identified Pma1, the H+-ATPase establishing the H+ gradient at the plasma membrane, as a central player of this control of TORC1 activity. As the transport activity of Pma1 itself is known to be stimulated by an increase of cytosolic protons, we suggest that Pma1 modulates TORC1 via signaling. Altogether, our results provide new insights on the central role of TORC1 in control of nutrient permeases in yeast.<br>Doctorat en Sciences<br>info:eu-repo/semantics/nonPublished
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Clingman, Carina C. "A Feedback Loop Couples Musashi-1 Activity to Omega-9 Fatty Acid Biosynthesis: A Dissertation." eScholarship@UMMS, 2014. http://escholarship.umassmed.edu/gsbs_diss/718.
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All living creatures change their gene expression program in response to nutrient availability and metabolic demands. Nutrients and metabolites can directly control transcription and activate second-‐messenger systems. In bacteria, metabolites also affect post-‐transcriptional regulatory mechanisms, but there are only a few isolated examples of this regulation in eukaryotes. Here, I present evidence that RNA-‐binding by the stem cell translation regulator Musashi-‐1 (MSI1) is allosterically inhibited by 18-‐22 carbon ω-‐9 monounsaturated fatty acids. The fatty acid binds to the N-‐terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. I identify stearoyl-‐CoA desaturase-‐1 as a MSI1 target, revealing a feedback loop between ω-‐9 fatty acid biosynthesis and MSI1 activity. To my knowledge, this is the first example of an RNA-‐binding protein directly regulated by fatty acid. This finding may represent one of the first examples of a potentially broad network connecting metabolism with post-‐transcriptional regulation.
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Keränen, Henrik. "Advances in Ligand Binding Predictions using Molecular Dynamics Simulations." Doctoral thesis, Uppsala universitet, Beräknings- och systembiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-230777.
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Biochemical processes all involve associations and dissociations of chemical entities. Understanding these is of substantial importance for many modern pharmaceutical applications. In this thesis, longstanding problems with regard to ligand binding are treated with computational methods, applied to proteins of key pharmaceutical importance. Homology modeling, docking, molecular dynamics simulations and free-energy calculations are used here for quantitative characterization of ligand binding to proteins. By combining computational tools, valuable contributions have been made for pharmaceutically relevant areas: a neglected tropical disease, an ion channel anti-drug-target, and GPCR drug-targets. We report three compounds inhibiting cruzain, the main cysteine protease of the protozoa causing Chagas’ disease. The compounds were found through an extensive virtual screening study and validated with experimental enzymatic assays. The compounds inhibit the enzyme in the μM-range and are therefore valuable in further lead optimization studies. A high-resolution crystal structure of the BRICHOS domain is reported, together with molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry studies. This work revealed a plausible mechanism for how the chaperone activity of the domain may operate. Rationalization of structure-activity relationships for a set of analogous blockers of the hERG potassium channel is given. A homology model of the ion channel was used for docking compounds and molecular dynamics simulations together with the linear interaction energy method employed for calculating the binding free-energies. The three-dimensional coordinates of two GPCRs, 5HT1B and 5HT2B, were derived from homology modeling and evaluated in the GPCR Dock 2013 assessment. Our models were in good correlation with the experimental structures and all of them placed among the top quarter of all models assessed. Finally, a computational method, based on molecular dynamics free-energy calculations, for performing alanine scanning was validated with the A2A adenosine receptor bound to either agonist or antagonist. The calculated binding free-energies were found to be in good agreement with experimental data and the method was subsequently extended to non-alanine mutations. With extensive experimental mutation data, this scheme is a valuable tool for quantitative understanding of ligand binding and can ultimately be used for structure-based drug design.
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Kirov, Miroslav [Verfasser], Lutz [Akademischer Betreuer] Schmitt, and Ulrich [Akademischer Betreuer] Schulte. "The incorporation of an unnatural amino acid to study the nucleotide binding domain of the ABC transporter HlyB from Escherichia coli / Miroslav Kirov. Gutachter: Ulrich Schulte. Betreuer: Lutz Schmitt." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2014. http://d-nb.info/1051076803/34.
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Huang, Charlie Chia Wei. "Regulation of Cat-1 gene transcription during physiological and pathological conditions." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1270242874.
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Wang, Haihong [Verfasser], Peter [Akademischer Betreuer] Illes, Wolfgang [Gutachter] Nörenberg, and Andreas [Gutachter] Reichenbach. "Amino acid residues constituting the agonist binding site of the human P2X3 receptor and subunit stoichiometry of heteromeric P2X2/3 and P2X2/6 receptors / Haihong Wang ; Gutachter: Wolfgang Nörenberg, Andreas Reichenbach ; Betreuer: Peter Illes." Leipzig : Universitätsbibliothek Leipzig, 2013. http://d-nb.info/1238366546/34.
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Zhang, Wei. "Directed Evolution of Glutathione Transferases with Altered Substrate Selectivity Profiles : A Laboratory Evolution Study Shedding Light on the Multidimensional Nature of Epistasis." Doctoral thesis, Uppsala universitet, Biokemi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-158400.
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Directed evolution is generally regarded as a useful approach in protein engineering. By subjecting members of a mutant library to the power of Darwinian evolution, desired protein properties are obtained. Numerous reports have appeared in the literature showing the success of tailoring proteins for various applications by this method. Is it a one-way track that protein practitioners can only learn from nature to enable more efficient protein engineering? A structure-and-mechanism-based approach, supplemented with the use of reduced amino acid alphabets, was proposed as a general means for semi-rational enzyme engineering. Using human GST A2-2*E, the most active human enzyme in the bioactivation of azathioprine, as a parental enzyme to test this approach, a L107G/L108D/F222H triple-point mutant of GST A2-2*E (thereafter designated as GDH) was discovered with 70-fold increased activity, approaching the upper limit of specific activity of the GST scaffold. The approach was further experimentally verified to be more successful than intuitively choosing active-site residues in proximity to the bound substrate for the improvement of enzyme performance. By constructing all intermediates along all putative mutational paths leading from GST A2-2*E to mutant GDH and assaying them with nine alternative substrates, the fitness landscapes were found to be “rugged” in differential fashions in substrate-activity space. The multidimensional fitness landscapes stemming from functional promiscuity can lead to alternative outcomes with enzymes optimized for other features than the selectable markers that were relevant at the origin of the evolutionary process. The results in this thesis suggest that in this manner an evolutionary response to changing environmental conditions can readily be mounted. In summary, the thesis demonstrates the attractive features of the structure-and-mechanism-based semi-rational directed evolution approach for optimizing enzyme performance. Moreover, the results gained from the studies show that laboratory evolution may refine our understanding of evolutionary process in nature.
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Oswal, Dhawal P. "Peroxisome proliferator-activated receptor alpha: Insight into the structure, function and energy homeostasis." Wright State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=wright1401279322.
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Nordesjö, Olle. "Searching for novel protein-protein specificities using a combined approach of sequence co-evolution and local structural equilibration." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-275040.
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Greater understanding of how we can use protein simulations and statistical characteristics of biomolecular interfaces as proxies for biological function will make manifest major advances in protein engineering. Here we show how to use calculated change in binding affinity and coevolutionary scores to predict the functional effect of mutations in the interface between a Histidine Kinase and a Response Regulator. These proteins participate in the Two-Component Regulatory system, a system for intracellular signalling found in bacteria. We find that both scores work as proxies for functional mutants and demonstrate a ~30 fold improvement in initial positive predictive value compared with choosing randomly from a sequence space of 160 000 variants in the top 20 mutants. We also demonstrate qualitative differences in the predictions of the two scores, primarily a tendency for the coevolutionary score to miss out on one class of functional mutants with enriched frequency of the amino acid threonine in one position.