Dissertations / Theses: 'Site-Directed Mutagenesi'

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Rúnarsdóttir, Saga. "Site-Directed Mutagenesis Studies of FucO." Thesis, Uppsala universitet, Institutionen för kemi - BMC, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-235136.

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Al-Khatib, Haifa Yousef. "Site Directed Mutagenesis of Dienelactone Hydrolase." Thesis, University of North Texas, 1995. https://digital.library.unt.edu/ark:/67531/metadc277940/.

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The clcD gene encoding dienelactone hydrolase (DLH) is part of the clc gene cluster for the utilization of the B-ketoadipate pathway intermediate chlorocatechol. The roles that individual amino acids residues play in catalysis and binding of the enzyme were investigated. Using PCR a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHi to ZscoRI subclone in the plasmid pUC19.

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Chen, Wei 1965. "Site Directed Mutagenesis Of Dienelactone Hydrolase." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc500900/.

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The role of individual amino acid residues of the enzyme dienelactone hydrolase was investigated. Using the polymerase chain reaction (PCR), a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHI to EcoRI clcD subclone in the plasmid pUC19. Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. Specifically modified proteins from mutated clcD genes (Arg 81 to alanine, Tyr 85 to phenylalanine and Arg 206 to alanine), were encoded by the mutant clones. Enzyme assays showed that dienelactone hydrolase activity of the mutants Arg 81 and Arg 206 was totally abolished. The DLHase enzyme activity of mutant Tyr 85 is greatly decreased by approximately two thirds.

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4

Marc, Daniel. "La myristylation de la proteine de capside vp4 du poliovirus; son role dans le cycle viral." Paris 7, 1991. http://www.theses.fr/1991PA077059.

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La proteine de capside vp4 du poliovirus, ainsi que des precurseurs vp0 et p1, est myristlee: sa glycine n-terminale est liee de maniere covalente a un acide gras tetra-decanoique. Cette modification co-traductionnelle est determinee par la sequence n-terminale de la proteine (gly#1ala#2gln#3val#4ser#5ser#6). Par mutagenese dirigee a l'aide d'oligonucleotides, nous avons modifie dans le cadn viral la sequence codant pour le signal de myristylation de la proteine. Des transcrits genomiques portant les differentes mutations ont ete synthetises in vitro, et leurs proprietes analysees in vitro par traduction en systeme acellulaire, et in vivo apres transfection de cellules de primates. La transfection du transcrit portant la mutation ser#5thr conduit a une production de virus possedant une proteine normalement myristylee. Toutes les autres mutations qui empechent totalement (gly#1arg et gly#1ala) ou partiellement (ser#5pro et ala#2pro) la myristylation de la proteine vp0 in vivo, abolissent l'infectivite des transcrits. Ces dernieres mutations n'empechent pas la replication des transcrits in vivo, mais affectent la maturation proteolytique du precurseur p1 in vitro. En outre, dans le cas des mutations de la glycine no 1, l'assemblage viral n'a pas lieu, le defaut se situant au niveau de l'assemblage des pentameres 14s. Dans le cas des deux autres mutations (ser#5pro et ala#2pro), des particules virales matures s'assemblent en quantite reduite, mais ces virons ne sont pas infectieux et semblent defectueux dans les etapes precoces de l'infection. La myristylation de la proteine vp4 et de ses precurseurs est donc indispensable au cycle viral. Elle joue role important au niveau de l'assemblage viral, mais semble egalement impliquee dans les etapes precoces de la decapsidation

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White, Malcolm F. "Yeast phosphoglycerate mutase studied by site-directed mutagenesis." Thesis, University of Edinburgh, 1989. http://hdl.handle.net/1842/24419.

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Wang, Xiaoshan. "Site-Directed Mutagenesis in Francisella Tularensis by Allelic." Thesis, Virginia Tech, 2007. http://hdl.handle.net/10919/36440.

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Francisella tularensis is a Gram-negative, facultative intracellular coccobacillus and the etiologic agent of tularemia for a wide variety of vertebrate and invertebrate animal species. Several species and subspecies of Francisella are currently recognized. However, the majority of infections are caused by F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B). Given the low infectious dose, multiple transmission routes, severity of illness, and lack of licensed vaccines, F. tularensis has long been considered a potential biological weapon and is now classified as a category A select agent by the National Institutes of Health and the Centers for Disease Control and Prevention.

The investigation of the mechanisms of pathogenesis by F. tularensis type A and B strains is hindered by the difficulty and lack of methods to mutate the putative genes that encode for virulence factors. New genetic tools have been developed that have enabled mutagenesis of F. tularensis type A and type B stains. However, site-specific mutations remain difficult to execute or these methods generate random mutations. In this study a novel method was developed to create site-directed mutations in a putative capsule biosynthesis locus to knock out encapsulation of the attenuated F. tularensis live vaccine strain. Two suicide vectors for mutagenesis of F. tularensis were constructed based on the commercial PCR cloning vector pSC-A. These vectors were created by inserting into the cloning site a kanamycin resistance gene boarded upstream by 1.3 kb of N-terminal DNA and downstream by 1.3 kb of C-terminal DNA that flanks the target gene. Cryotransformation was used to introduce the vectors into F. tularensis. Open reading frame (ORF) FTT0793, which may encode for an ABC transporter involved in capsule export, was initially selected for mutagenesis in order to generate a mutant that was nonencapsulated, but could still synthesize capsule and induce a host immune response. Mutagenesis of this gene was successful. However, phenotypic assays could not confirm that the mutant was nonencapsulated compared to the parent. Therefore, adjacent ORFs FTT0798 and FTT0799, which may encode for a galactosyl transferase and mannosyl transferase, respectively, were also deleted to completely knock out capsule synthesis. The resulting mutant appeared to be nonencapsulated as determined by negative staining transmission electron microscopy.

In this study, a plasmid and method for generating allelic exchange mutants is reported, which should be useful for generating additional mutants of F. tularensis for use in clarifing the roles of specific genes. This vector is currently being used to make a nonencapsulated mutant of a virulent type A strain to determine the role of capsule in virulence.

Master of Science

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Bowker-Kinley, Melissa M. "Pyruvate dehydrogenase kinase Kinetics, site-directed mutagenesis, and regulation /." [Bloomington, Ind.] : Indiana University, 2005. http://wwwlib.umi.com/dissertations/fullcit/3183930.

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Thesis (Ph.D.)--Indiana University, 2005.
Source: Dissertation Abstracts International, Volume: 66-07, Section: B, page: 3690. Chair: Robert A. Harris. Title from dissertation home page (viewed Oct. 5, 2006).

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Tito, Donald. "Site-directed mutagenesis of hydrogenase genes in Azotobacter chroococcum." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=56889.

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Accessory hydrogen uptake genes have been identified in a region of the Azotobacter chroococcum genome about 5 kb downstream of the hydrogenase structural genes (hupSL). DNA sequencing has revealed six genes (hupABYCDE) in this region. These genes are probably transcribed in the same direction as hupSL but are probably in a different operon. Mutational analysis had shown that disruption of the hupB, hupY, hupD and hupE genes gives a Hup$ sp-$ phenotype. In the present work additional mutational analysis, using Tn5, a Tn5 -derivative containing a promoterless lacZ gene, and a kanamycin resistance gene, confirms the direction of transcription and the separate nature of the hupABYCDE operon, and extends the region known to be necessary for Hup activity to hupA and possibly to 1.6 kb upstream of hupA.

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Owegi, Margaret. "Site-directed mutagenesis of yeast V-ATPase subunit d." Virtual Press, 2005. http://liblink.bsu.edu/uhtbin/catkey/1319550.

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V-ATPases are enzymes found in all eukaryotic cells. They are organized into a peripheral membrane complex (V1) and an integral membrane complex (V0). VI is responsible for ATP hydrolysis and generates the energy used by Vo to pump protons from the cytosol into the vacuole. Subunit d is a component of Vo possibly located at the interface between V 1 and V. in the V-ATPase complex. We hypothesize that subunit d could be involved in the structural and functional coupling of VI and Vo. This was tested by generating point mutations along the open reading frame of subunit d from yeast. The mutations F94A, H128A, D173A, D217A, D261A, E317A, W325A, E328A and C329A, all in conserved regions of the protein sequence, were characterized by examining their growth phenotype and by assessing their ATPase specific activity, proton transport and V1Vo assembly in purified vacuolar membranes. The mutations E317A, W325A, E328A and C329A had reduced ATPase and proton transport activities. In addition, V1Vo assembly was compromised by the mutation W325A. Our results suggest that residues at the carboxyl-end of subunit d are important for ATPase activity, proton pumping and V1Vo assembly at the membrane.
Department of Chemistry

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10

Schmidt, William Richard. "Site-directed mutagenesis of the ncd microtubule motor protein." Thesis, This resource online, 1996. http://scholar.lib.vt.edu/theses/available/etd-12302008-063348/.

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Dou, Chao. "Site-directed mutagenesis as probes for F₁-ATPase function /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1997. http://wwwlib.umi.com/cr/ucsd/fullcit?p9812499.

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12

Patel, Jigar. "Site directed mutagenesis of L-pyrrolysine in dimethylamine methyltransferase." Connect to resource, 2009. http://hdl.handle.net/1811/36534.

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Merry, Stephen Alan Paul. "Exciton transfer and trapping in photosystem II." Thesis, Imperial College London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286500.

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CRIVELLO, PIETRO. "New molecular insights into HLA immunogenicity." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2012. http://hdl.handle.net/10281/29854.

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Alloreactivity is the major barrier to solid organ and hematopoietic stem cell transplantation (HSCT), determining important clinical events such as graft rejection and graft versus host disease. This biological phenomenon is due to the immunogenicity of allogeneic Human Leukocyte Antigens (HLA) expressed in transplanted tissues and recognized by T cell receptor (TCR) on the surface of alloreactive T cells. In the past, the hosting laboratory identified an immunogenic T cell epitope (TCE) encoded by a subset of HLA-DPB1 alleles, thereby defining permissive and non-permissive DPB1 mismatches between donor and recipient associated with different clinical outcomes after HSCT. Aim of this thesis is to elucidate the molecular basis underlying the different clinical impact of DPB1 mismatches in order to improve our understanding of HLA immunogenicity in the context of transplantation. To achieve this aim, we investigated if the clinical effect of DPB1 mismatches was reflected by the strength of their in vitro alloreactive T cell response and, subsequently, we characterized the alloreactive response to one of the most immunogenic DPB1 alleles, HLA-DPB1*09:01 (HLA-DP9), at the molecular level by homology modeling driven site directed mutagenesis. The strength of the alloreactive T cell response to HLA-DP was assessed in Mixed Lymphocyte Reactions between healthy responder/stimulator pairs mismatched only at the DPB1 locus, followed by quantitative assessment of responder T cells upregulating the cell surface activation marker CD137. We observed significantly higher frequencies of activated T cells against non-permissive mismatches (n=9; mean 10.13% ± 7.51%) compared to permissive mismatches (n=15; mean 2.34% ± 2.82% p<0.05). SDM of HLA-DP9 was performed on 6 polymorphic amino acid residues predicted to be crucial for interaction with bound peptide (positions 9, 35, 55, 69, 76 and 84), and on 2 amino acid residues putatively involved in direct interaction with the TCR (positions 56 and 57). These residues were mutated into amino acids naturally occurring in other HLA-DP variants. A lentiviral vector expression platform was used to express the wild type or mutant HLA-DP in 2 HLA homozygous reporter B lymphoblastoid Cell lines. A panel of 8 alloreactive T cell effectors, specific for wild type HLA-DPB1*09:01 (n=6) or for DPB1*10:01 and DPB1*1701, respectively, but crossreactive to DPB1*09:01 (n=2), was used to evaluate the impact of mutagenesis on T cell recognition by gIFN ELISpot or CD107a degranulation assay. For T cells specifically alloreactive to HLA-DPB1*09:01, recognition was influenced by a complex pattern of residues, which was different for each of the 6 effectors studied. In contrast, for the 2 T cells cross-reactive to HLA-DPB1*09:01, only 2 amino acids (positions 69 and 76) had an influence on allorecognition, suggesting the presence of a more restricted set of T cell epitopes in the context of cross-reactivity rather than nominal specificity. These results are consistent with the peptide involvement in alloreactivity to HLA-DPB1*09:01, and underline the complexity of allorecognition which may be one of the mechanisms underlying the immunogenicity of this molecule. Finally, an additional collaborative study with the North Italian Program for Solid Organ Transplantation was aimed at predicting the functional role of amino acid point mutations in newly described naturally occurring variants of HLA-A3 and A32, respectively, by homology modeling. These studies confirmed data from the literature suggesting a relevant putative role of amino acid substitutions at positions 114 and 116, present in a variant of HLA-A3, on the molecular shape and charge of the peptide binding groove. In contrast, point mutations at position 151 and 258 found in two variants of HLA-A32 and A3, respectively, were not predicted to have important functional impacts, due to limited structural or biochemical changes of the groove. In conclusion, our results shed new light on the molecular basis of T cell alloreactivity. In particular, we showed that the stronger immunogenicity of non-permissive DPB1 mismatches is reflected by a divergent and complex T cell epitope repertoire with a putative involvement of the peptide repertoire presented to the alloreactive TCRs. Identification of specific allopeptides associated with these responses will allow to further dissect their molecular basis.

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Aronica, Pietro. "Development of a new protocol for computatinal site-directed mutagenesis." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/40433.

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Mutagenesis, the technique of mutating individual amino acids on proteins and peptides, is an important part of protein engineering and analysis. By changing residues and measuring the effect of the mutation on the properties of the protein such as its structure and interaction, a deeper understanding can be gained, which can be used to design new, better biomolecules. However, when performed experimentally, mutagenesis can be expensive, time-consuming and a rate-limiting step in research. Computational tools can be used to aid within this context, but a review of existing methods revealed gaps in the current literature. The Parasol Protocol was developed in order to address these issues and provide a new method that would be suitable for virtual scans and which relied on molecular dynamics. The Parasol Protocol is a tool which utilises the AMBER package framework to mutate at will between any pair of natural amino acids, incorporating a wide range of possible different functional groups and transformations. It is cheap, quick and easy to use while still allowing a high degree of control. After the development, work focused on validating the protocol by applying it to various test cases. Experimentally observed interactions and structures were compared with those obtained via computational simulations, performed using the Parasol Protocol. Our understanding of those systems has deepened thanks to these studies and in some cases it had remarkable agreement with laboratory results, indicating predictive power. We think that the Parasol Protocol has performed well so far and could become a standard method used in molecular dynamics and protein design.

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Lloyd, John S. "Heterologous expression and site-directed mutagenesis of soluble methane monooxygenase." Thesis, University of Warwick, 1997. http://wrap.warwick.ac.uk/59600/.

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The purpose of this investigation was to study the heterologous expression of soluble methane monooxygenase (sMMO) genes from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. Using the T7-RNA polymerase expression system, the entire sMMO operon and subclones (constructed using the polymerase chain reaction) were over-expressed in E. coli. Results obtained using the Me. capsulatus (Bath) sMMO operon confirmed previous reports (C. West, G. P. C. Salmond, H. Dalton and 1. C. Murrell (1992). J Gen. Microbial. 138, 1301-1307) that functional expression of protein B and the reductase occurred but the hydroxylase was inactive. Similar results were obtained by expressing the sMMO operon of Ms. trichosporium OB3b in E. coli, using plasmids previously described (D. Jahng and T. K. Wood (1994). Appl. Environ. Microbiol. 60, 2473-2482). Protein B, the reductase and orfY were over-expressed and purified from E. coli using glutathione-Stransferase fusion proteins and affinity chromatography. The expression of sMMO genes from Mc. capsulatus (Bath) and Ms. trichosporium OB3b was studied in Pseudomonas putida. A previous report (D. Jahng and T. K. Wood (1994). Appl. Environ. Microbial. 60, 2473-2482) had suggested that functional expression of sMMO from Ms. trichosporium OB3b was achieved in P. putida Fl. Attempts to repeat this work proved that protein B and the reductase were functionally expressed, but the hydroxylase was inactive. Similar results were obtained for the heterologous expression of the sMMO operon from Mc. capsulatus (Bath) in P. putida. Methanotrophs were used for the heterologous expression of sMMO via two strategies. (1) The expression of sMMO from Mc. capsulatus (Bath) and Ms. trichosporium OB3b was studied in Methylomonas album B08 and Methylocystis parvus OBBP. These are methanotrophs that do not express sMMO, but express particulate MMO (pMMO) only, to utilise methane as a sole carbon and energy source. Functional expression of the sMMO operon of Ms. trichosporium OB3b was achieved in Mm. album BG8, however, recombinant sMMO enzyme activity was poor and problems were encountered with the growth of the sMMO positive transconjugant methanotrophs. (2) sMMO-minus marker exchange mutants of Ms. trichosporium OB3b (H. Martin and 1. C. Murrell (1995). FEMS Microbiol. Letts. 127, 243-248) were complemented with plasmid encoded genes and functional sMMO expression was obtained. Southern hybridisation analysis revealed that the plasmid DNA had integrated into the chromosome of the Ms. trichosporium OB3b sMMO-minus mutant via a single homologous recombination event between the mmoX genes. Protein B from Mc. capsulatus (Bath) is inactivated by proteolysis to give rise to a truncated form designated protein B'. The Met 12-Gly 13 cleavage site was modified by site-directed mutagenesis to Met12-Gln13 which improved the stability of the protein when incubated at room temperature. Only after prolonged incubation was protein B' formed. Recombinant protein B from Ms. trichosporium OB3b also appears to be unstable, and readily degraded when incubated at room temperature. The cleavage of protein B to inactive protein B' may be a general regulatory mechanism that occurs within the cell to regulate sMMO activity.

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Chitpinityol, Supannee. "Heterologous expression and site-directed mutagenesis of the enzyme chymosin." Thesis, University of Reading, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320101.

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18

Farr, George William. "Site-directed mutagenesis of beta tubulin's putative GTP-binding domain." Case Western Reserve University School of Graduate Studies / OhioLINK, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=case1060367517.

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19

Jack, Richard F. "Characterization and site-directed mutagenesis of NifU from Azotobacter vinelandii." Diss., This resource online, 1995. http://scholar.lib.vt.edu/theses/available/etd-10042006-143858/.

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Blom, Lillemor. "Investigation of the interactions between the bacterial homologue to actin, and the chaperone GroEL/ES through a combination of protein engineering and spectroscopy." Thesis, Linköping University, Department of Physics, Chemistry and Biology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-15818.

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Molecular chaperones help many proteins in the cell reach their native conformation. The mechanism with which they do this has been studied extensively, but has not been entirely elucidated. This work is a continuation of the study done by Laila Villebeck et al. (2007) on the conformational rearrangements in the eukaryotic protein actin in interaction with the eukaryotic chaperone TRiC. In this study the intentions were to analyze the protein MreB, a prokaryotic homologue to actin, when interacting with the prokaryotic chaperone GroEL. The purpose was to investigate if the mechanisms of GroEL and TRiC are similar. The analysis of the conformation of MreB was to be made through calculations of fluorescence resonance energy transfer (FRET) between two positions in MreB labeled with fluorescein. A MreB mutant was made through site-specific mutagenesis to enable labeling at a specific position. Another single mutant and a corresponding double mutant needed for these measurements were avaliable from earlier studies. The results from fluorescence measurements on these mutants indicated that the degree of labeling was insufficient for accurate determination of FRET. Suggestions are made on improvements of the experimental approach for future studies.

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Jonnalagadda, Madhuri. "Site Directed Mutagensis of Bacteriophage HK639 and Identification of Its Integration Site." TopSCHOLAR®, 2008. http://digitalcommons.wku.edu/theses/42.

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Bacteriophages affect bacterial evolution, pathogenesis and global nutrient cycling. They are also the most numerous and diverse group of biological entities on the planet [1, 2, 3, 4, 5, 6]. Members of the Lambda phage family share a similar genetic organization and control early gene expression by suppressing transcription, a process known as antitermination. Transcription antitermination in Lambda is mediated by a phage-encoded protein whereas in lambdoid phage HK022, antitermination is mediated by a phage-encoded RNA molecules. Recent results suggest that another bacteriophage called HK639 also appears to use RNA-mediated antitermination. To characterize this newly identified phage we generated site directed mutations and identified where the phage integrates into the chromosome of its bacterial host.

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Mizoguchi, Tadashi Jack Gray Harry B. Gray Harry B. Richards Jack. "Probing the role of the active-site Cysteine of Azurin by site-directed mutagenesis /." Diss., Pasadena, Calif. : California Institute of Technology, 1996. http://resolver.caltech.edu/CaltechETD:etd-01162009-111204.

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De, Villiers Jacques Izak. "Mutagenesis studies of a glycoside hydrolase family 2 enzyme." Thesis, Stellenbosch : Stellenbosch University, 2015. http://hdl.handle.net/10019.1/98110.

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Thesis (MSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Galactooligosaccharides are produced by the transglycosylation activity of β-galactosidases (β-gal, EC 3.2.1.23) when utilising lactose as a substrate. They have emerged as important constituents used in the food and pharmaceutical industries owing to their prebiotic properties. Although transglycosylation was discovered in 1951 (Wallenfels 1951), and a number of β-gals have had their transglycosylation activity characterised, the activities of these enzymes are not optimal for industrial use. Their tendency to favour the hydrolytic reaction over the transglycosylation reaction, coupled with the production of shorter chain oligosaccharides has driven scientists to investigate altering protein structure both to increase chain lengths and the amount of oligosaccharide produced at lower substrate concentrations. In an attempt to alter the amount of oligosaccharide produced by a metagenomically derived β-gal belonging to the glycosyl hydrolase 2 family, random and site-directed mutagenesis were used. A randomly mutagenised library was screened on SOB agar plates containing 5% (w/v) lactose which should select for clones that synthesise oligosaccharides at relatively low concentrations. No such activity was detected. Site-directed mutagenesis was also utilised to alter protein structure. It was confirmed that the β-gal utilised in this study belonged to the glycosyl hydrolase 2 family through mutation of the predicted catalytic acid/base glutamic acid to a non-catalytic residue, thus removing activity. Another mutation was utilised to investigate if it was possible to increase the degree of polymerisation of oligosaccharides produced by the β-gal. This mutation was successful in increasing the degree of polymerisation. Biochemical characterisation of the β-gal revealed that it exhibited optimal activity at pH 8.0, with a temperature optimum of 30°C. The β-gal exhibited a Km and Vmax of 54.23 mM and 2.26 μmol/minute-1/mg protein-1 respectively, similar to kinetic parameters that have been determined for a number of previously characterised enzymes.
AFRIKAANSE OPSOMMING: Galaktooligosakkariede word geproduseer deur die transglikosileering aktiwiteit van β-galaktosidase (β-gal, EG 3.2.1.23) wanneer hulle laktose as 'n substraat gebruik. Hierdie oligosakkariede het na vore gekom as 'n belangrike bestandeel vir gebruik in die voedsel en farmaseutiese bedryf as gevolg van hulle prebiotiese eienskappe. Alhoewel transglycosylation al in 1951 ontdek is (Wallenfels 1951) en 'n aantal β-gals se transglycosylation aktiwiteit gekenmerk is, is hierdie ensieme nie ideaal vir industriële toepassings nie. Die geneigdheid om die hidrolitiese reaksie oor die transglycosylation reaksie bevoordeel, tesame met die produksie van korter oligosakkariede het wetenskaplikes ondersoek genoop om die proteïenstruktuur te verander om ketting-lengte en die kwantiteit van oligosakkaried geproduseer teen laer substraat konsentrasies te verhoog. In 'n poging om die opbrengs van die oligosakkaried wat deur 'n metagenomiese β-gal wat aan die glycosyl hidrolase 2 familie behoort te verander, is lukraak en terrein gerigte-mutagenese gebruik. Die mutagenese biblioteek is op SOB agarplate met 5% (w/v) lactose gekeur, om klone wat die fenotipe wat verband hou met die produksie oligosakkaried teen relatiewe lae konsentrasies te selekteer. Geen aktiwiteit is opgemerk nie. Terrein gerigte-mutagenese is ook gebruik om die proteïenstruktuur te verander. Deur ‘n bioinformatiese voorspelling, is dit bevestig dat die β-gal wat in hiedie studie gebruik word tot die glycosyl hidrolase 2 familie behoort. Dit is gedoen deur mutasie van die voorspelde katalitiese suur/basis glutamiensuur na 'n nie-katalitiese oorskot, dus die verwydering van aktiwiteit. Nog ‘n mutasie is gebruik om te ondersoek of dit moontlik was om die ketting-lengte van die oligosakkaried wat deur die β-gal geproduseer is te verhoog. Die mutasie was suksesvol in die verhoging van die oligosakkaried wat geproduseer was. Biochemiese karakterisering van die β-gal het getoon dat hierdie β-gal optimale aktiwiteit het by pH 8.0, met 'n optimum temperatuur van 30°C. Die β-gal het 'n Km en Vmax van 54.23 mM en 2.26 μmol/minute-1/mg proteïen-1 onderskeidelik, soortgelyk aan kinetiese parameters wat bepaal word vir ensieme wat voorheen gekenmerk is.

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Daniell, Sarah Jane. "Site-directed mutagenesis of the rat Dâ†2(Long) dopamine receptor." Thesis, University of Kent, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241618.

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Fan, Yan Baranger Anne M. Katzenellenbogen John A. Zhao Huimin Silverman Scott K. "Exploring protein-RNA interactions with site-directed mutagenesis and phage display." Urbana, IL.: University of Illinois, 2009. http://hdl.handle.net/2142/14755.

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Eriksson, Sollenberg Ulla. "Characterization of galanin receptors using chimeric peptides and site-directed mutagenesis /." Stockholm : Department of Neurochemistry, Stockholm University, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-37259.

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Diss. (sammanfattning) Stockholm : Stockholms universitet, 2010.
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Härtill 4 uppsatser.

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Kaylor, Joanna Jacelyn. "Studies of the aggregation of [alpha]-synuclein using site-directed mutagenesis /." Diss., Digital Dissertations Database. Restricted to UC campuses, 2004. http://uclibs.org/PID/11984.

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Butland, Gareth Paul. "Expression and site-directed mutagenesis of a bacterial nitric oxide reductase." Thesis, University of East Anglia, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327513.

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Man, Wai Jin. "Studies on the mechanism of citrate synthase using site directed mutagenesis." Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386673.

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Woodcock, Sarah Catherine. "Studies on the mechanism of porphobilinogen deaminase using site directed mutagenesis." Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.385454.

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31

Carter, P. J. "Site-directed mutagenesis of the tyrosyl tRNA synthetase of Bacillus stearothermophilus." Thesis, University of Cambridge, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372646.

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32

Burgess, J. G. "Construction of a system for site directed mutagenesis in Rhodobacter sphaeroides." Thesis, Imperial College London, 1990. http://hdl.handle.net/10044/1/47793.

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33

Smith, Thomas John. "β-lactamase of Staphylococcus aureus PC1 : studies using site-directed mutagenesis." Thesis, University of Edinburgh, 1992. http://hdl.handle.net/1842/11411.

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A system was developed for generating site-directed mutants of the β-lactamase of Staphylococcus aureus PC1, and for expression of the mutant proteins in S.aureus. The mutant genes were made in vitro using the polymerase catalysed chain reaction (PCR), by the overlap extension method (Ho, S.N. et al. [1989] Gene 77, 51-59). The were cloned in Escherichia coli and transferred to S.aureus using an E.coli-S.aureus shuttle vector. The mutant proteins were, like the wild-type, secreted into the medium by the transformed S.aureus cells. It was found that high-level constitutive expression, at a level that was possible for the wild-type-β-lactamase, was not obtainable with the three mutants that were studied in this work. Mutations of the β-lactamase Shine-Dalgarno sequence, which originally occurred spontaneously, were exploited to enable the mutant genes to be established in S.aureus at a reduced level of expression. The S70A mutation (amino acid numbering is according to Ambler, R.P. et al. [1991] Biochemical Journal 276, 269-270) was designed to abolish catalysis whilst preserving binding of the substrate. My co-workers have crystallised the native mutant enzyme and obtained a high-resolution electron density map. Work is under way to obtain similar data for complexes between the mutant enzyme and substrates or inhibitors. The S130A mutant has diminished activity, but k_cat for ampicillin and benzylpenicillin is affected considerably more than for cephaloridine and nitrocefin. The steady-state kinetic properties of the A238S mutant are not greatly altered, although the mutant is more strongly inhibited by cefotaxime than the wild-type is. It was observed that a 0.84kb HindIII-XbaI fragment, containing part of the staphylococcal β-lactamase gene, is unstable in E.coli when cloned in silation from the rest of the gene. All the clones that were characterised had undergone changes that would reduce expression of the truncated β-lactamase gene; a large proportion of these had mutations of the promoter region. It is proposed that expression of the truncated gene is a strong selective disadvantage to E.coli, although the intact gene is essentially harmless.

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34

Caffrey, Michael Stephen. "Characterization of cytochrome c structure and function by site-directed mutagenesis." Diss., The University of Arizona, 1991. http://hdl.handle.net/10150/185346.

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A dual Rhodobacter capsulatus/Rhodobacter sphaeroides genetic system has been used to study the structure and function of R. capsulatus cytochrome c₂. In the first part of this study, the processing, stability and in vivo, functionality of nine site-directed mutants have been examined. Mutations were designed to test various structural and functional properties of cytochrome c₂ such as redox potential (Y75C, Y75F and Y75S), surface charges (K12D, K14E, K32E and K14E/K32E), and protein conformation (P35A and W67Y). All R. capsulatus cytochrome c₂ mutants, except Y75C and Y75S, were overproduced in both R. capsulatus and R. sphaeroides suggesting that these mutations had no effects on heme attachment and protein stability. Furthermore, all R. capsulatus cytochrome c₂ mutants transcomplement for photosynthetic growth a cytochrome c₂ minus mutant of R. sphaeroides suggesting that these mutations function in vivo. Analysis of the spectroscopic, redox potential, kinetic and stability properties of mutants Y75C and Y75F suggested that R. capsulatus tyrosine 75 or its equivalent in other species plays an important role in formation of a hydrogen bonding network which results in maintaining redox potentials and stability of cytochromes c in general. It was found that the charge mutants exhibited small reductions in redox potentials that were consistent with the substitution of positively charged groups with negatively charged groups. Kinetic analyses of the charge mutant photooxidations by R. sphaeroides reaction centers suggested that the lysine groups surrounding the cytochrome c exposed heme edge are not critical to cytochrome c structure and function but play a role in optimal molecular orientation for electron transfer reactions. In addition, denaturation studies of the charge mutants indicated that lysine groups in the amino terminal alpha helix may be important to cytochrome c₂ stability. Analysis of the spectroscopic, redox potential, kinetic and stability properties of mutants P35A and W67Y suggested that proline 35 and tryptophan 67 of R. capsulatus cytochrome c₂ or their equivalents in other species are important to stability but not critical to the structure, redox potential, and function of cytochromes c in general.

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35

Setterquist, Robert Alan. "Site-directed mutagenesis of the nitrogenase MoFe protein from Azotobacter vinelandii." Thesis, Virginia Polytechnic Institute and State University, 1989. http://hdl.handle.net/10919/50091.

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A model describing the potential amino acid ligands to the four 4Fe-4S centers (P-clusters) within the Azotobacter vinelandii nitrogenase MoFe protein is presented. Based on interspecies and intersubunit amino acid comparisons of the α- and ß-subunits of the MoFe protein, and the FeMoco biosynthetic proteins, NifE and NifN, four conserved residues (Cys62, His83, Cys88, Cys154 all proposed P-cluster ligands) within the α- subunit were targeted for site-directed mutagencsis studies. In order to define a range of acceptable substitutions, 35 specific site-mutants have been constructed, each with a different amino acid replacement at one of the four targeted positions. Previous studies indicated that these residues were important for MoFe activity, and may act as metallocenter ligands. Unusual redox and spectroscopic properties of the Fe-S centers suggest the involvement of ligands other than the four typical cysteines, though extrusion requirements indicate that some thiol ligands are likely. Surprisingly, mutants with an Asp, Gly, Thr, or Ser substituted for Cys88 are still capable of diazotrophic growth (Nif+), though whole cell and crude extract acetylene reduction activity is lowered. Several substitutions (Cys, Asp, Phe, Asn, Met, Tyr, Leu) are tolerated at the His83 position, these Nif+ mutant strains also have varying acetylene reduction rates and growth rates. All mutants with substitutions at positions 62, 154, resulted in complete loss of diazotrophic growth. The results could be interpreted by the following explanations: 1) Our proposed model for the P-cluster ligation within the MoFe protein is incorrect. 2) Some substitutions permit P-cluster rearrangement to a semi-functional state. 3) Either, P-clusters are not absolutely essential for diazotrophic growth, or the enzyme can function with a reduced number of these metal centers.
Master of Science
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36

Khaja, Sara. "Site-Directed Mutagenesis in Citrus paradisi Flavonol-Specific 3-O-Glucosyltransferase." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etd/2453.

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Flavonoids are plant secondary metabolites that have significant biochemical and physiological roles. Biosynthesis of these compounds involves several modifications, most predominantly glucosylation, which is catalyzed by glucosyltransferases (GTs). A signature amino acid sequence, the PSPG box, is used to identify putative clones and has been shown to be involved in UDP-glucose binding. Site-directed mutagenesis is used to answer questions regarding the structure and function of this family of enzymes, particularly what allows some GTs to be more selective towards some substrates than others. The grapefruit (Citrus paradisi) flavonol-3-O-glucosyltransferase (CpF3GT) is specific for flavonol substrates and will not glucosylate anthocyanidins. Comparison of the CpF3GT sequence with that of Vitis vinifera GT, which glucosylates both flavonols and anthocyanidins, provided the basis for the amino acid substitution of proline 145, alanine 374, and alanine 375 in CpF3GT to threonine, aspartate, and glycine, respectively, to test the affect on GT’s affinity for flavonoid substrates.

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37

Duncan, Tammi Rae. "Site-directed mutagenesis of the SPOR domain from Escherichia Coli FtsN." Thesis, University of Iowa, 2011. https://ir.uiowa.edu/etd/954.

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Escherichia coli cell division is a complex process involving over 30 proteins that are recruited to the division site. One of the important division proteins is FtsN, which has a C-terminal peptidoglycan (PG) binding region called the SPOR domain. Most SPOR domain proteins are probably involved in bacterial cell division, but their precise role in this process is not known. Although the structure of the FtsN SPOR domain has been solved by NMR, nothing is known about how the domain binds PG. Understanding the SPOR:PG interaction is important because it could lead to novel insights into PG metabolism during cell division. We hypothesize that the SPOR domain from FtsN recognizes and binds septal PG. To test this hypothesis we have conducted a comprehensive mutagenesis of the FtsN SPOR domain to identify amino acid residues critical for septal localization and PG binding. We targeted 33 residues and made a total of 92 point mutants, all of which were tested for septal localization. Our results revealed four amino acids that are critical for septal localization. All four of these residues map in or near the â-sheet, which we now propose is the PG binding site. Further analysis of localization-defective proteins in a PG binding assay led to the realization that the assay measures nonspecific binding to bulk PG rather then specific binding to septal PG. An important priority for the future will be to develop a better PG binding assay.

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38

Sheng, Mei. "Site Directed Mutagenesis of β-Ketoadipate Succinyl-Coenzyme A Transferase II from Acinetobacter Calcoaceticus." Thesis, University of North Texas, 1993. https://digital.library.unt.edu/ark:/67531/metadc279281/.

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The role of specific amino acid residues in β-ketoadipate succinyl-coenzyme A transferase II from Acinetobacter calcoaceticus was investigated. A 1412 base pair BamiHI-EcoRI fragment carrying the catIJ genes was amplified by polymerase chain reaction and inserted into pUCl9 to generate the plasmid pCATEl9. Escherichia coli DH5α (pCATEl9) carrying only the catlJ genes expressed 3-fold higher enzyme activity than the parent strain. Two mutants were constructed by site directed mutagenesis so that glutamate was replaced by a glutamine at positions Gln155 and Gln193 in the ß subunit of the primary amino acid sequence of the CoA transferase. Both mutants produced transferase that was catalytically active suggesting that Glu155 and Glu193 do not participate directly in catalysis.

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39

De, Nagornoff Leititia. "Site-directed mutagenesis of active-site residues in the hydroxylase component of soluble methane monooxygenase." Thesis, University of Warwick, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.439651.

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40

陳家輝 and Ka-fai Joseph Chan. "Production of novel biological proteins by hybridoma technique and site directed mutagenesis." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1993. http://hub.hku.hk/bib/B31211148.

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41

Chan, Ka-fai Joseph. "Production of novel biological proteins by hybridoma technique and site directed mutagenesis /." [Hong Kong] : University of Hong Kong, 1993. http://sunzi.lib.hku.hk/hkuto/record.jsp?B1364130X.

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42

Temperley, Richard James. "Generation of a reporter for mitochondrial gene expression studies." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.369782.

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43

Gossner, Anton Gerhard. "Bio-engineering and genetic manipulation of ovine interleukin-2." Thesis, University of the West of England, Bristol, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297877.

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44

Shi, Qingli. "Expression and site-directed mutagenesis studies of a ribosome-inactivating protein : neo-trichosanthin." HKBU Institutional Repository, 1999. http://repository.hkbu.edu.hk/etd_ra/221.

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45

Arnett, Diana. "Site-Directed Mutagenesis of the -127 Activator Binding Site of the qa-2 Gene of Neurospora crassa." Youngstown State University / OhioLINK, 2000. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1004634030.

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46

Delavari, Azar. "Structure-activity investigation on laccases by computational and site directed mutagenesis studies." Doctoral thesis, Universitat Politècnica de Catalunya, 2016. http://hdl.handle.net/10803/404452.

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Laccases belong to multi copper oxidase enzyme family (EC 1.10.3.2). Their capacity to oxidíze a wide range of substrates makes them very attractive for the industry and are growing in importance for environmentally-friendly synthesis. Laccases have three different copper sites including, type 1 (T1), type 2 (T2) and type 3 (T3). The function of the T1 site is shuttling electrons from the substrate to the trinuclear copper cluster. During the catalytic cycle of laccase, four electrons are removed from four substrate molecules, which are finally transferred to reduce oxygen to two water molecules .Comparison of the kinetic parameters using several laccases and several substrates reveals that the reaction rate of laccase correlates with the redox potential difference between the T1 copper and the substrate. In recent years, the demonstrated potential of laccases in a range of applications has motivated the progress of laccase engineering efforts. Computational simulations can reveal targets for protein engineering to be explored by site-directed mutagenesis (or semi-rational approaches). In this work we used computational methods for studying interaction of different substrates with laccases and structural activity of the enzyme. The goal of the present study was to characterize the laccase binding pocket of fungal and bacterial laccases in order to establish their common pharmacophoríc characteristics. For this purpose, we first performed molecular docking studies to identify those residues involved in the interaction with diverse substrates. Our results indicate that bacterial laccase {1UVW) has less hydrophobic and aromatic residues in the activity site in comparison to other fugal structures of this study, as a result, find a pose that interacts with residues needs more energy. Subsequently, we evaluated the effect of protonation state of a conserved residue in fungal laccase, Asp/Glu, through molecular dynamics simulation. In a subsequent step, we applied QMMM-2QM-MD approach for one of the fungal laccase structure (3FU8) for calculating redox potential value. The result indicates that the difference in redox potentials changes from 7-17 to 74-92 kJ/mol if the redox state of T1Cu and DMP in the other subunit change and we correctly predict that CuT1ox/DMPred state is more stable than the CuT1red/DMPox state. After the insight gathered from computational studies we started site directed mutagenesis studies on two residues of the binding pocket in order to find their effect on the redox potential value. We made a combinatorial library for position 192 and 296 in MtlL T2. The clone contained A192P and L296W (3H12) mutation and clone contained A192P and 296L {19G8) showed activity with violuríc acid 1.23 and 1.33 fold higher than parental type, respectively. Moreover, the clone contained A192R and L296W (15H11) and clone with mutation A192R and L296L {5B4) showed higher activity with molybdenum compound in comparison to parental type. After experimental characterization of the 19G8 and 5B4 mutants, we studied the structural changes produced in the binding pocket. For this purpose we generated a three-dimensional structure of the two mutants using M.albomices laccase as template by homology modelling. Whereas the former mutant exhibits a similar binding pocket to the template, the latter appears to be smaller. In any case, subsequent docking studies did not show any differential behaviour and ligands could bind to both binding pockets in a similar way. Finally, we calculated the redox potential of the mutant A296L MaL that is similar to the former mutant, yielding a value of 167 kJ/mol. This is higher than the value obtained for MalL supporting the effect of this mutation on the redox potential.
Las lacasas pertenecen a la familia de enzimas multícobre oxidadas (EC 1.10.3.2). Su capacidad de oxidar una amplia gama de sustratos las hace muy atractivas para la industria y su utilización está creciendo en importancia para la síntesis respetuosa del medio ambiente. Las lacasas tienen tres tipos diferentes de cobre: tipo 1 (T1), Tipo 2 (T2) y típo 3 (T3). La función del sitio de T1 es la de transportar electrones desde el sustrato al clúster de cobre trinuclear. Durante el ciclo catalítico de la lacasa, cuatro electrones son transferidos desde cuatro moléculas de sustrato para reducir oxígeno a dos moléculas de agua. La comparación de los parámetros cinéticos utilizando varias lacasas y varios sustratos revela que la velocidad de reacción de la lacasa se correlaciona con la diferencia de potencial redox entre el cobre T1 y el sustrato. En los últimos años, el potencial demostrado por las lacasas en una gama de aplicaciones ha motivado el progreso en la ingeniería de lacasas. Las simulaciones computacionales pueden revelar residuos clave que pueden ser cambiados por mutagénesís dirigida (o enfoques semi-racionales). En este trabajo se han utilizado métodos computacionales para el estudio de la interacción de diferentes sustratos con lacasas y ver su efecto sobre la actividad. El objetivo del presente estudio fue caracterizar la unión de lacasa bolsillo de lacasas fúngícas y bacterianas con el fin de establecer sus características farmacofórícas comunes. Para este propósito, hemos realizado estudios de anclaje moleculares para identificar aquellos residuos que participan en la interacción con diversos substratos. Nuestros resultados indican que la lacasa bacteriana (1UVN) tiene un número menor de residuos hidrófobos y aromáticos que las estructuras fúngicas, como consecuencia la unión no es tan fuerte. Posteriormente, se evaluó el efecto del estado de protonación de un residuo Asp / Glu conservado en lacasas fúngicas a través de dinámica molecular. En una etapa posterior, se aplicó enfoque QMMM-2QM-MD para uno de la estructura lacasa fúngica (3FU8) para calcular el valor potencial redox. El resultado índica que la diferencia en los potenciales redox cambios 7-17 a 74-92 kJ/mol sí el estado redox de T1Cu y DMP en la otra subunidad cambio y correctamente predecir qué estado CuT1ox / DMPred es más estable que el CuT1red / estado DMPox. Después de los estudios computacionales se llevó a cabo un estudio de mutagénesis dirigida sobre dos residuos del bolsillo de unión, con el fin de encontrar su efecto sobre el valor potencial redox. Con este objetivo se llevó a cabo una biblioteca combinatoria para la posición 192 y 296 en MtL T2. El clan contenía A192P y L296W (3H 12) y el clan contenía la mutación A192P y L296L (19G8) mostraron una actividad con ácido violurico 1,23 y 1,33 veces mayor que la de tipo parental, respectivamente. Por otra parte, el clon contenía A192R y L296W (15h11) y el clon con A192R mutación y L296L (5B4) mostraron una mayor actividad con el compuesto de molibdeno en comparación con el tipo parental. Después de la caracterización experimental de los mutantes 19G8 y 5B4, estudiamos los cambios estructurales que se producen en el bolsillo de unión. Con este fin generamos una estructura tridimensional de los dos mutantes utilizando la lacasa de M.albomices como plantilla, por medio de la modelización por homología. Mientras que el primer mutante exhibe un bolsillo de unión similar al de la plantilla, éste es más pequeño en el segundo mutante. En cualquier caso, los estudios de anclaje molecular posteriores no mostraron ningún comportamiento diferencial y los ligandos podrían unirse a los dos bolsillos de unión de una manera similar. Finalmente, se calculó el potencial redox de la mutante A296L MaL que es similar al mutante 19G8, obteniéndose un valor de 167 kJ/mol. Este valor es más alto que el obtenido para MaL, apoyando el efecto que tiene esta mutacíón sobre el potencial redox.

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47

Taylor, John Michael. "Revealing flagellar motor protein interactions by site directed mutagenesis in R. sphaeroides." Thesis, Nottingham Trent University, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.444661.

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48

Gartland, Martin John. "Site-directed mutagenesis of the aromatic amino acid aminotransferase of Escherichia coli." Thesis, University of Nottingham, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293014.

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49

Liu, Hsiao-Hui. "EcoRV endonuclease studied by site-directed mutagenesis and oligodeoxynucleotides containing modified bases." Thesis, University of Newcastle Upon Tyne, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.287807.

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50

Wang, Xing-Guo. "Alteration of substrate specificity in clostridial glutamate dehydrogenase by site-directed mutagenesis." Thesis, University of Sheffield, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387762.

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