Dissertations / Theses on the topic 'Substrate-binding proteins'
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Hendry, Garth S. "Dependence of substrate-water binding on protein and inorganic cofactors of photosystem II /." View thesis entry in Australian Digital Theses Program, 2002. http://thesis.anu.edu.au/public/adt-ANU20041124.140348/index.html.
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Fesser, Stephanie Marion. "Contribution of RNA binding proteins to substrate specificity in small RNA biogenesis." Diss., Ludwig-Maximilians-Universität München, 2013. http://nbn-resolving.de/urn:nbn:de:bvb:19-173105.
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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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Sarma, Ranjana. "Investigations of nucleotide-dependent electron transfer and substrate binding in nitrogen fixation and chlorophyll biosynthesis." Thesis, Montana State University, 2009. http://etd.lib.montana.edu/etd/2009/sarma/SarmaR1209.pdf.
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The studies presented in this thesis include studies of nucleotide-dependent conformations of the electron donor protein in nitrogenase and dark-operative protochlorophyllide reductase (DPOR) characterized using small-angle x-ray scattering and x-ray diffraction methods. Nitrogen fixation and chlorophyll synthesis are involved in the reduction of high energy bonds under physiological conditions. Both make use of elegant reaction mechanisms made possible by complex enzyme systems which are evolutionarily related. Nitrogenase reduces nitrogen to ammonia and is a two-component metalloenzyme composed of Fe protein and MoFe protein. For nitrogen reduction, the Fe protein and MoFe protein associate and dissociate in a manner concomitant with hydrolysis of at least two MgATP molecules and enables the concomitant transfer of at least one electron from Fe protein to MoFe protein. During chlorophyll biosysnthesis, the rate limiting step is catalyzed by a two-component metalloenzyme called DPOR. The two components of DPOR are BchL and BchNB proteins and these share high level of sequence similarity with the Fe protein and the MoFe protein, respectively. Based on this sequence similarity and biochemical data available, it is proposed that the reaction mechanism is similar to nitrogenase mechanism in which the components of DPOR associate and dissociate in a nucleotide dependent manner, to enable intercomponent electron transfer. Fe protein and BchL present as unique examples of proteins that couple nucleotide dependent conformational change to enable electron transfer for high energy bond reduction. The present studies have been directed at studying the low resolution studies of MgATP-bound wild-type Fe protein and its comparison to the structure of the proposed mimic, i.e, L127 Delta Fe protein. The studies presented show evidence of the MgATP-bound wild-type Fe protein having a conformation very different from the L127 Delta Fe protein. The chapters also include detailed characterization of the structure of BchL in both MgADP bound and nucleotide-free states which offer detailed insights in the structure based mechanism of BchL, with primary focus on identifying key residues involved in componenet docking and in electron transfer. Together, the studies on the Fe protein and BchL have furthered our understanding of mechanism of electron transfer in these complex enzyme systems.
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Jaya, Nomalie Naomi. "SUBSTRATE BINDING SITE FLEXIBILITY OF SMALL HEAT SHOCK PROTEINS AND FACTORS CONTRIBUTING TO EFFICIENT CHAPERONE ACTIVITY." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/193550.
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sHSPs maintain partially denaturing substrates in a soluble sHSP-substrate complex. The heterogeneous interaction between sHSPs and substrate within the complex has prevented a detailed study of the mechanism of sHSP substrate protection. Here, purified sHSPs and heat sensitive substrates were used to investigate the mechanism of sHSP chaperone action. Results presented provide new insights into how sHSPs recognize substrates, the architecture of the sHSP-substrate complex and factors contributing to chaperone efficiency.Direct evidence defining the role of the sHSP N-terminal arm and alpha crystallin domain in sHSP-substrate interactions is limited. A photoactivatable probe was site- specifically incorporated into PsHsp18.1, and cross-linking to substrate in sHSP-substrate complexes was quantified. The structurally flexible N-terminal arm of PsHsp18.1 makes strong contacts with both substrates tested, however differences in interaction were seen in the conserved alpha crystallin domain. Regions on the sHSP showing the strongest cross-links to substrates are buried within the dodecamer, supporting the model that the sHSP oligomer undergoes rearrangement or dissociation prior to substrate interactions.The arrangement of sHSPs and substrates whithin the complex is poorly defined. Limited proteolysis and chemical modification was combined with mass spectrometry to probe the sHSP-substrate complex using multiple sHSPs and substrates. This analysis reveals that a similar partially-denatured form of substrate is protected in the complex irrespective of sHSP identity. Further, sHSP in the complex is protected from proteolysis for a longer time compared to free sHSP. These data suggest that sHSPs and substrate are distributed both internally and on the periphery of the sHSP-substrate complex.Exact properties of the sHSP N-terminal arm contributing to protection are poorly defined. A molecular dynamics (MD) study was designed to test the hypothesis that the N-terminal arm could assume multiple conformations that can readily interact with denaturing substrates. Preliminary data suggest that at increased temperatures amino acids in the N-terminal arm form specific clusters which could act as substrate interaction sites. MD simulations, mutagenesis and altering the kinetics of substrate aggregation suggest that the conformational space occupied by the N-terminal arm at increased temperatures, along with flexibility and rate of substrate aggregation contribute to differences in chaperone efficiency.
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Hendry, Garth S., and Garth Hendry@baldwins com. "Dependence of substrate-water binding on protein and inorganic cofactors of photosystem II." The Australian National University. Research School of Biological Sciences, 2002. http://thesis.anu.edu.au./public/adt-ANU20041124.140348.
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The photosynthetic water oxidation reaction is catalyzed by an inorganic Mn4OxCaClyHCO3-z cluster at the heart of the oxygen evolving complex (OEC) in photosystem II. In the absence of an atomic resolution crystal structure, the precise molecular organization of the OEC remains unresolved. Accordingly, the role of the protein and inorganic cofactors of PSII (Ca2+, HCO3- and Cl-) in the mechanism of O2-evolution await clarification. In this study, rapid 18O-isotope exchange measurements were applied to monitor the substrate-water binding kinetics as a function of the intermediate S-states of the catalytic site (i.e. S3, S2 and S1) in Triton X-100 solubilized membrane preparations that are enriched in photosystem II activity and are routinely used to evaluate cofactor requirements. Consistent with the previous determinations of the 18O exchange behavior in thylakoids, the initial 18O exchange measurements of native PSII membranes at m/e = 34 (which is sensitive to the 16O18O product) show that the fast and slowly exchanging substrate-waters are bound to the catalytic site in the S3 state, immediately prior to O2 release. Although the slowly exchanging water is bound throughout the entire S-state cycle, the kinetics of the fast exchanging water remains too fast in the S2, S1 [and S0] states to be resolved using the current instrumentation, and left open the possibility that the second substrate-water only binds to the active site after the formation of the S3 state. Presented is the first direct evidence to show that fast exchanging water is already bound to the OEC in the S2 state. Rapid 18O-isotope exchange measurements for Ex-depleted PSII (depleted of the 17- and 23-kDa extrinsic proteins) in the S2 state reveals a resolvable fast kinetic component of 34k2 = 120 ± 14 s-1. The slowing down of the fast phase kinetics is discussed in terms of increased water permeation and the effect on the local dielectric following removal of the extrinsic subunits. In addition, the first direct evidence to show the involvement of calcium in substrate-water binding is also presented. Strontium replacement of the OEC Ca2+-site reveals a factor of ~3-4 increase in the 18O exchange of the slowly exchanging water across the S3, S2 and S1 states while the kinetics of the fast exchanging water remain unchanged. Finally, a re-investigation of the proposed role for bicarbonate as an oxidizable electron donor to photosystem II was unable to discern any 18O enrichment of the photosynthetically evolved O2 in the presence of 18O-bicarbonate. A working model for O2-evolution in terms of these results is presented.
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Fesser, Stephanie Marion [Verfasser], and Klaus [Akademischer Betreuer] Förstemann. "Contribution of RNA binding proteins to substrate specificity in small RNA biogenesis / Stephanie Marion Fesser. Betreuer: Klaus Förstemann." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2013. http://d-nb.info/1055907793/34.
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Yuan, Ming. "Antiphagocytosis by Yersinia pseudotuberculosis : role of the YopH target proteins." Doctoral thesis, Umeå : Umeå University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-957.
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Wisniewska, Magdalena. "Biochemical studies on IGF and IGF-binding proteins interactions & structural investigations on the SH3 domain of Crk-associated tyrosine kinase substrate p130cas (CAS)." [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=978198549.
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Escobedo, Pascual Albert. "Structural Insights into Substrate Binding and Regulation of E3 Ubiquitin Ligases in the Nedd4 Family using NMR Spectroscopy." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/284605.
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Nedd4L is a HECT-type E3 ubiquitin ligase (it covalently binds ubiquitin molecules before transferring them to the final substrate). Ubiquitination is a posttranslational modification (PTM) that labels proteins for a variety of fates, the most relevant one being proteasome-mediated degradation. Nedd4L is responsible for the regulation of the turnover of the sodium channel ß-ENaC as well as Smad2/3, mediator proteins of the signalling pathway activated by TGF-ß-like cytokines. It also targets the TGF-ß receptor itself. Defects in its function have been related to hereditary hypertension (Liddle’s syndrome), and could be relevant in certain sorts of cancer and metastasis.
CDK8/9 and GSK3-ß are two kinases that regulate the phosphorylation of the Smads, enabling them to carry out their function in cooperation with transcription factors and other partner proteins. At the same time, they label the Smads for their recognition by ubiquitin ligases. This provides the cell with a mechanism to give a transient response to the cytokines of the TGF-ß type. In order to identify the residues and the phosphorylation patterns that are relevant for the interactions of the Smads with both the transcription factors and the ubiquitin ligases, we have prepared a set of phosphopeptides corresponding to the sequences of Smad1 and Smad3.
Like all other members of the Nedd4 family, Nedd4L has a multi-domain architecture of the type C2-WW-HECT. Several ligases of the family exist in a latent conformation established through inter-domain contacts that occlude the catalytic site in the HECT domain, involving either the C2 domain (Smurf1, Smurf2, WWP2, Nedd4, Nedd4L) or the central segment where the WW domains are located (Itch). Certain cellular events displace these contacts, inducing the transition to the active conformation. In the case of Nedd4L, increases of the intracellular levels of Ca2+ activate the ligase. The hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) delivers into the cytosol the inositol 1,4,5-triphosphate (IP3), a second messenger that mobilizes the intracellular Ca2+ reserves. The C2 domain of Nedd4L interacts both with Ca2+ ions and with IP3. Using a structural and biophysical approach based on Nuclear Magnetic Resonance (NMR) we have described the specific interactions between the HECT and C2 domains that inhibit the catalytic function. Ca2+ binds the C2 domain with high affinity using the same binding surface and compromises these contacts. In addition, it mediates the interaction with IP3. These results provide the structural fundament for the activation and the relocation to the plasma membrane of Nedd4L mediated by Ca2+.
The HECT domain has a highly conserved PY site (HECT-PY). The PY motifs are the sequences recognized by WW domains. Central to this recognition is the coordination of the tyrosine residue in the PY motif by the WW domain. In the crystallographic structure of the Nedd4L HECT domain the tyrosine residue of the HECT-PY motif appears buried in the hydrophobic core and not accessible for binding. It has been shown that the WW domains of Nedd4L recognize the HECT-PY motif of the ligase only after the unfolding of the HECT domain. We raised the hypothesis that the recognition of the HECT-PY motif by one of Nedd4L WW domains may play a role in the auto-ubiquitination mechanism of the ligase. Our data confirm that only when the fold of the HECT domain is partially damaged, the PY site is accessible for being recognized by the WW domains. We present the NMR solution structure of the complex between the WW3 domain and the HECT-PY motif. The site is protected in functional Nedd4L molecules, which are able to recognize it in damaged molecules and label them with ubiquitin for degradation.Nedd4L és una E3 ubiquitín lligasa responsable de la regulació de la vida mitja del canal de sodi ß-ENaC i de Smad2/3, proteïnes mediadores de la ruta de senyalització activada per citocines TGF-ß. Defectes en la seva funció han estat relacionats amb la hipertensió hereditària (Síndrome de Liddle), i podrien ser rellevants en determinats tipus de càncer i metàstasi. CDK8/9 i GSK3-ß són dues quinases que regulen l’estat de fosforilació de les Smads, habilitant-les per dur a terme llur funció en cooperació amb factors de transcripció al mateix temps que les marquen per ser reconegudes per ubiquitín lligases. Amb l’objectiu d’identificar els residus i els patrons de fosforilació rellevants hem preparat un set de fosfopèptids que corresponen a les seqüències de Smad1/3. Nedd4L presenta una arquitectura multi-domini C2-WW-HECT. Diverses lligases de la família de Nedd4 existeixen en una conformació latent en què contactes inter-domini oclouen el lloc catalític en el domini HECT, involucrant bé el domini C2 (Smurf1/2, WWP2, Nedd4, Nedd4L) o la zona central amb els dominis WW (Itch). Certs esdeveniments cel•lulars desplacen aquests contactes, induint la transició a la conformació activa. L’increment dels nivells intracel•lulars de Ca2+ activa Nedd4L. La hidròlisi del fosfolípid de membrana PIP2 allibera l’IP3 provocant aquest increment. El domini C2 de Nedd4L interacciona tant amb el Ca2+ com amb l’IP3. Utilitzant l’RMN hem descrit els contactes HECT-C2 en la conformació latent i hem observat que el Ca2+ s’uneix al domini C2 amb alta afinitat utilitzant el mateix lloc d’unió, a més d’afavorir la interacció amb l’IP3. Així, hem aportat el fonament estructural per a l’activació i relocalització a la membrana cel•lular de Nedd4L. El domini HECT presenta un lloc PY altament conservat (HECT-PY). Els motius PY són reconeguts pels dominis WW. Proposem que el reconeixement del motiu HECT-PY per part d’un dels dominis WW de Nedd4L estigui implicat en l’auto-ubiquitinació. Hem observat que només quan el plegament del domini HECT està compromès, el lloc PY és accessible. Presentem l’estructura per RMN del complex WW3-HECT-PY. El motiu està protegit en molècules funcionals de Nedd4L, capaces de reconèixer-lo en molècules danyades i ubiquitinar-les.
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Dabrowski, Christian. "Mutagenesis of the substrate binding site of protein disulfide isomerase." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66879.
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Protein disulfide isomerase (PDI) is the most abundant and best characterized member of the extensive PDI family of proteins found in the endoplasmic reticulum. PDI plays an important role in protein folding. It is composed of four thioredoxin-like domains denoted as a-b-b'-a'. Recent studies have identified a hydrophobic surface on the b' domain as the main binding site for the unfolded protein substrates of PDI. Here, the main hydrophobic residues of the b' domain were mutated in a bb' construct and tested for their ability to bind to the 14-residue mastoparan peptide. Most of the mutants had a significant effect on the binding affinity. In reverse experiments the mastoparan peptide was mutated and NMR titrations used to determine the orientation of the peptide on the bb' binding surface. Finally, the catalytic a and a' domains were individually tested for binding to unfolded RNaseA by NMR. Neither catalytic domain exhibited any direct interaction with the unfolded substrate.La protéine disulfure isomérase (PDI) est le membre le mieux connu et caractérisé de la nombreuse famille de PDI qui se trouve dans le réticulum endoplasmique. PDI joue un rôle important dans le repliement des chaînes protéiques. Elle est composée de quatre domaines désignés a-b-b'-a' de type thiorédoxine. Des études récentes ont identifié une surface hydrophobe dans le domaine b' comme étant le lieu principal d'interaction avec les substrats déroulés de la PDI. Ici, les résidus hydrophobes principaux du domaine b' ont été mutés et testés pour leur interaction avec un peptide de quatorze résidus nommé mastoparan. La plupart des mutants ont eu un impact important sur l'affinité de la PDI pour son substrat. Dans des expériences contraires où le peptide mastoparan a été muté, des titrations par RMN ont révélé l'orientation du peptide sur la surface d'interaction du domaine b'. Finalement, les domaines catalytiques a et a' ont été individuellement testés par RMN pour leur affinité envers l'ARNase A déroulée. Les deux domaines n'ont montré aucune interaction avec le substrat déroulé.
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Farah, Sahar. "Identification of Rho-associated protein kinaseà as an insulin receptor substrate-1 binding protein." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ28422.pdf.
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Morris, Benjamin L. "Understanding and targeting the C-terminal Binding Protein (CtBP) substrate-binding domain for cancer therapeutic development." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4434.
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Cancer involves the dysregulated proliferation and growth of cells throughout the body. C-terminal binding proteins (CtBP) 1 and 2 are transcriptional co-regulators upregulated in several cancers, including breast, colorectal, and ovarian tumors. CtBPs drive oncogenic properties, including migration, invasion, proliferation, and survival, in part through repression of tumor suppressor genes. CtBPs encode an intrinsic dehydrogenase activity, utilizing intracellular NADH concentrations and the substrate 4-methylthio-2-oxobutyric acid (MTOB), to regulate the recruitment of transcriptional regulatory complexes. High levels of MTOB inhibit CtBP dehydrogenase function and induce cytotoxicity among cancer cells in a CtBP-dependent manner. While encouraging, a good therapeutic would utilize >100-fold lower concentrations. Therefore, we endeavored to design better CtBP-specific therapeutics. The best of these drugs, 3-Cl and 4-Cl HIPP, exhibit nanomolar enzymatic inhibition and micromolar cytotoxicity and showed that CtBP enzymatic function is subject to allosteric interactions. Additionally, the function of the substrate-binding domain has yet to be examined in context of CtBP’s oncogenic activity. To this end, we created several point mutations in the CtBP substrate-binding pocket and determined key residues for CtBP’s enzymatic activity. We found that a conserved tryptophan in the catalytic domain is imperative for function and unique to CtBPs among dehydrogenases. Knowledge of this and other residues allows the directed synthesis of drugs with increased potency and higher CtBP specificity. Early work interrogated the importance of these residues in cell migration. Taken together, this work addresses the utility of the CtBP substrate-binding domain as a target for cancer therapeutics.
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Farah, Sahar. "Identification of Rho-associated protein kinase-alpha as an insulin receptor substrate-1 binding protein." Thesis, University of Ottawa (Canada), 1998. http://hdl.handle.net/10393/4152.
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Insulin receptor substrate-1 (IRS-1) is phosphorylated on multiple tyrosine residues by ligand-activated insulin receptor. These tyrosine phosphorylation sites serve to dock several SH2-containing signaling proteins. In addition, IRS-1 also contains several protein modules that have been implicated in protein-protein or protein-lipid interactions. In an attempt to identify novel proteins that may interact with these IRS-1 protein modules, yeast two-hybrid screening was employed. The bait, corresponding to the N-terminal 500 amino acids of the Xenopus IRS-1 (XIRS-1), was comprised of a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain and a SAIN (Shc and IRS-1 NPXY binding) domain. Screening of a Xenopus oocyte cDNA library with the bait resulted in the isolation of a partial Xenopus cDNA, XROK$\alpha$. The cloned cDNA contains an open reading frame of about 500 amino acids (in frame with the N-terminal GAL4 activating domain) which are 90% identical to the C-terminus of the recently identified RhoA-associated protein kinase $\alpha$ (ROK$\alpha$). The partial XROK$\alpha$ cDNA contains the putative Rho binding domain as well as the C-terminal pleckstrin homology/cysteine rich domain (PH/CRD) but lacks the N-terminal serine/threonine kinase domain. Using the yeast two-hybrid system, we showed that XROK$\alpha$ interacts strongly with a constitutively active form of RhoA (V14-RhoA). Further analysis indicated that the XIRS-1 PTB domain is specifically involved in binding to XROK$\alpha$. To further characterize the potential role of XROK$\alpha$ in insulin signalling, I have cloned the entire coding sequence of XROK$\alpha$ by a combination of hybridyzation screening and PCR amplification. I have also generated XROK$\alpha$ specific polyclonal antibodies. Using these antibodies I have detected endogenous XROK $\alpha$ protein by both Western-blotting and by immune kinase assay in vitro. Taken together, these studies have identified an IRS-1 binding serine/threonine kinase which may play an important role in modulating insulin signalling.
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Chen, Ce-Belle. "The role of mannose-binding protein-associated serine proteases in complement activation : interactions with mannose-binding protein and mode of substrate recognition." Thesis, University of Oxford, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400560.
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Fischer, Marcus. "Structural biophysics of ligand, fragment and water interactions with substrate binding protein SiaP." Thesis, University of York, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.542843.
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Knight, James D. R. "The Structural and Functional Identity of the Protein Kinase Superfamily." Thesis, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/20232.
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The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.
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Court, Naomi Wynne. "The subcellular localisation, tissue expression, substrate specificity and binding partners of stress-activated protein kinase-3." University of Western Australia. School of Biomedical and Chemical Sciences, 2004. http://theses.library.uwa.edu.au/adt-WU2004.0084.
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[Truncated abstract] Cells need to be able to detect changes in their surrounding environment and transduce these signals into the appropriate cellular compartments. One of the major ways that the cell achieves this signal transduction is through the process of phosphorylation. Protein kinases are the enzymes responsible for catalysing this transfer of phosphate groups from ATP to amino acid residues of their specific substrates. A subfamily of serine/threonine kinases known as the Mitogen-Activated Protein Kinases (MAPKs) is essential in a diverse range of cell processes including growth, metabolism, differentiation and death. The first identified MAPKs, the Extracellular Signal-Regulated Kinases (ERKs), were found to be activated in response to mitogenic stimuli such as growth factors. However, since the discovery of the ERKs, other pathways leading to the activation of related kinases have been recognised. These kinases are preferentially activated in response to stress, and are thus termed “Stress-Activated Protein Kinases” or SAPKs. They consist of the c-Jun N-terminal kinase isoforms 1, 2 and 3 (also called SAPK1γ, SAPK1α and SAPKβ respectively) and the p38 MAPKs, p38α, p38β, p38γ and p38δ (also called SAPK2a, SAPK2b, SAPK3 and SAPK4 respectively). A major challenge in this field has been to identify the substrates and functions of the SAPKs. This has been partly achieved by the development of inhibitors for the JNK MAPKs and SAPK2a/b. However, no inhibitors currently exist that specifically inhibit SAPK3 and SAPK4. Therefore, elucidating the function of these SAPKs has proved more difficult. Recent studies suggest that SAPK3 may play a unique role in the cell compared to other members of the p38 MAPK family. For example, several signalling proteins appear to specifically activate SAPK3 in certain circumstances while not activating other members of the p38 MAPK family. In addition, SAPK3 contains a unique sequence motif that allows it to bind to specialised domains known as PDZ domains. The interaction of SAPK3 with proteins containing these domains may regulate its subcellular localisation and interactions with other proteins in the cell. This project was undertaken to expand the knowledge on the expression, localisation, substrate specificity and binding partners of SAPK3. In Chapter 3 of this thesis, a SAPK3 monoclonal antibody was evaluated for its ability to specifically recognise endogenous SAPK3 protein. SAPK3 was found to be expressed in immortalised cell lines and primary cultures of neonatal rat myocytes, and to be colocalised with the mitochondria of these cells. This co-localisation remained unaltered in response to treatment with the nuclear export inhibitor Leptomycin B, and with exposure to osmotic shock, suggesting that SAPK3 substrates may be localised at the mitochondria
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Wagner, Judith Nastjenka. "The HSP100 protein ClpA: Significance of the N-terminal domain in substrate recognition and binding and characterization of the novel substrate RepE." [S.l. : s.n.], 2007. http://nbn-resolving.de/urn:nbn:de:bsz:25-opus-60188.
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Walter, Julia [Verfasser]. "The Autopalmitoylated Vesicular Transport Protein Bet3 : Biochemical and Fluorescence-based Characterization of Membrane and Substrate Binding / Julia Walter." Berlin : Freie Universität Berlin, 2018. http://d-nb.info/1155761081/34.
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Wang, Fen. "In silico and in vitro determination of substrate specificity for Breast Cancer Resistance Protein (BCRP) transporter at the blood-brain barrier." Thesis, Uppsala universitet, Institutionen för farmaci, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-444527.
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Background The Breast Cancer Resistance Protein (BCRP) drug transporter is important for drug disposition and plays a critical role in regulating drug entry into the brain. Its substrate spectrum overlaps with substrates of Multi Drug Resistance Protein 1 (MDR1, P-gp), which influences and complicates the interpretation of data on drug distribution into tissues (e.g. brain). Distinguishing BCRP mediated transport from the transport by the MDR1 is often problematic. However, with new in vitro tools, this is now possible. In this project, two drug compounds, i.e. Dantrolene and Ritonavir, were investigated using these new in vitro models. The results from the experimental in vitro assay were matched with molecular dynamics (MD) simulations. Using coarse-grained (CG) simulations, a model of the BCRP transporter in a lipid bilayer was built, this model is based on the human BCRP structure revealed by Taylor et al (2017). Simulations were run for Dantrolene (a known substrate of BCRP) independently three times, and another with Ritonavir (a non-substrate) three times. Aim To determine substrate specificity for the BCRP transporter for two compounds, and to construct a CG model of BCRP transporter to see whether in silico methods can be used as an alternative for assessing substrate specificity. Methods Madin-Darby canine kidney (MDCK) II cell line with no endogenous canine MDR1 (cMDR1) expression (MDCKcMDR1-KO), overexpressing human MDR1 (hMDR1) (MDCK-hMDR1cMDR1-KO) and stable expression of human BCRP (hBCRP) (MDCK-hBCRPcMDR1-KO) cells were cultured and used in Transwell experiments. Samples were analyzed using LC-MS/MS to determine the substrate concentrations. Apparent permeability and efflux ratio was calculated and evaluated. MD simulations used the Martini 3 CG force field, and were run with Gromacs (version 2020.4). Tools including MODELLER, INSANE and others were used to construct the initial model (Webster, 2000; Wassenaar et al., 2015), for parameterization of substrate and non-substrate molecules. And visual inspection was done with the visual molecular dynamics (VMD) program and PyMOL. Results In vitro transport experiment confirmed that Dantrolene is a BCRP specific substrate, and Ritonavir is MDR1 specific substrate. Following simulations of these two compounds, Dantrolene is observed to stay in the transmembrane domains (TMD) for a certain period (on average several hundreds of nanoseconds), while Ritonavir is not found to bind in the TMD, which provides a proof of concept for future studies.
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Petrovic, Dusan [Verfasser], Birgit [Akademischer Betreuer] Strodel, Christel M. [Gutachter] Marian, and Johannes [Gutachter] Kästner. "Computational Enzyme Evolution and Design: Studies of Protein Dynamics and Substrate Binding / Dusan Petrovic ; Gutachter: Christel M. Marian, Johannes Kästner ; Betreuer: Birgit Strodel." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2018. http://d-nb.info/1163108065/34.
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Sukumar, Preethi. "Molecular dissection of substrate and inhibitor binding to the D-galactose-H⁺ symport protein (GalP) from Escherichia coli - the bacterial homologue of GLUT1." Thesis, University of Leeds, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.713697.
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The D-galactose-H+ symporter, Ga1P, of Escherichia coil was subjected to mutagenesis to elucidate the molecular mechanism of substrate and ligand recognition, to define the roles of individual amino acid residues and to stabilize the protein in a conformation favorable for crystallization. Twenty-five mutants were constructed during the course of this study using the following four criteria: 1) elimination of protease susceptible sites; 2) sequence similarities and differences to close homologues; 3) residues based on previous mutagenesis in GLUT1 that might lock the protein in a conformation suitable for crystallization; and 4) residues putatively involved in electrostatic interactions. Almost all mutant proteins were shown to be expressed and inserted into the membrane at levels equivalent to the WT-GaIP(His)6, as verified by SDS-PAGE and Western blot. A combination of different criteria such as ability to transport sugars and bind high affinity inhibitory ligands, cytochalasin B and forskolin, enabled comparison between the WT- and GalP(His)6 mutants. Analyses of these mutants allowed for the following important observations. (1) Peptide-mass fingerprinting indicated that the outer membrane protease cleaves between Arg455 and Lys456 in the C-terminal end of GalP(His)6. Neutral substitution of these residues did not eliminate the C-terminal truncation, indicating that the adjacent residues might also serve as substrates for the outer-membrane proteases. However, the purified proteins of R455T and K456A mutants were observed to migrate as a single species, with the C-terminal protein not co-purifying with the full-length protein. Additionally, initial crystallization trials of K456A-GalP(His)6 appears promising. (2) Neutral substitution of Asp312 (D3 12G) severely impaired sugar-transport while retaining the ability to bind ligands. Furthermore an additional occluded-ligand binding state was observed shifting the equilibrium of the mutant towards the inward-facing conformation in the absence of substrate. This inwardly locked mutant can serve as a good candidate for crystallization. (3) When the Tyr273 residue (N-terminal end of TMH7) was substituted with Ile, the ability to transport D-glucose and D-galactose was severely impaired. Moreover, cytochalasin B affinity was completely lost and the affinity for forskolin reduced, indicating that this residue might be directly involved in sugar and cytochalasin B recognition. The Pro368 residue substitution by Gln drastically reduced D-glucose recognition while maintaining the affinity to D-galactose, indicating that Pro368 might participate in the discrimination of the C-4 hydroxyl group on sugar substrates. (4) Mutation of G1u438, Thr439 and Lys440 of the 'VPETIC' motif at the end of TMH12 indicated that these residues are likely to be involved in maintaining the overall conformation of the transporter through electrostatic interactions rather than having a direct involvement in substrate and ligand binding. Additional mutants not mentioned here were found to have either no effect or minor perturbations in either the structure or helical packing of the transporter indicating that they were not essential for the overall transport process.
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Janke, Kirsten [Verfasser], Eric [Akademischer Betreuer] Metzen, and Verena [Akademischer Betreuer] Jendrossek. "Characterisation of apoptosis-stimulating p53 binding protein 2 (ASPP2) as a new substrate for asparaginyl hydroxylation / Kirsten Janke. Gutachter: Verena Jendrossek. Betreuer: Eric Metzen." Duisburg, 2013. http://d-nb.info/1042373507/34.
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Sanford, Brianne. "Role of Coupled Dynamics and a Strictly Conserved Lysine Residue in the Function of Bacterial Prolyl-tRNA Synthetase and Substrate Binding by a Related trans-Editing Enzyme ProXp-ala." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397645941.
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Subramaniam, Srisunder. "Studies of conformational changes and dynamics accompanying substrate recognition, allostery and catalysis in bacteriophage lambda integrase." The Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1111655332.
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Chakrabarti, Kalyan Sundar. "Solution Structural Studies And Substrate Binding Properties Of The Amino-Terminal Domain Of E.coli Pantothenate Synthetase." Thesis, 2008. http://hdl.handle.net/2005/1103.
Full textAbstract:
Pantothenate synthetase (PS), which catalyzes the last step in the pantothenate (vitamin B5) biosynthesis, is a dimeric enzyme present in bacteria, fungi and plants. The enzymatic properties of PS from Escherichia Coli, Mycobacterium tuberculosi, Fusarium Oxysporum, Lotus japonicus, Oryza sativum, Brassica napus and Arabidopsis thaliana have been characterized. The chemical reaction and the proposed mechanism of reaction are identical for PS, irrespective of the source. However, from an enzyme mechanistic point of view, plant PS’s are dissimilar to their bacterial counterparts, in that they exhibit “allosteric behavior”, a property that has not been observed in the bacterial enzyme. The behavior is quite remarkable when one takes into consideration the fact that plant PS’s share a high degree of sequence identity (~ 40%) with the bacterial enzymes. Even more intriguing is the structural mechanism proposed to explain the observed differences in structure between the PS’s from E.Coli and M.tb, which share a 42% sequence identity. Till date there is no structural information available on the plant PS’s and of the substrate bound conformation of E.coli PS. This thesis aims to provide an understanding on some aspects of the structure – function relationship of this physiologically important enzyme. Specifically, the solution properties of E. coli PS have been examined using high-resolution multinuclear, multidimensional NMR methods. Given the large size of the full-length protein (~ 63 KDa), the structurally distinct N and C-terminal domains were cloned and expressed as individual proteins and their properties investigated.
Towards this end, the tertiary fold of the 40 kDa dimeric amino-terminal domain of E. coli pantothenate synthetase has been determined using multidimensional multinuclear nuclear magnetic resonance (NMR) methods (PDB entry 2k6c). Sequence specific resonance assignments for backbone HN, 15N, 13Cα, 13C', sidechain 13Cβ and aliphatic 13CH3 (of isoleucine, leucine and valine residues) were obtained using perdeuterated ILV-methyl protonated samples (BMRB entry 6940). Secondary structure of nPS was determined from 13C secondary chemical shifts and from short and medium range NOEs. Global fold of the 40 kDa homo-dimeric nPS has been determined using a total of 1012 NOEs, 696 dihedral angles, 260 RDCs, 155 hydrogen bonds, radius of gyration potential, non-crystallographic symmetry potential and database derived potential based upon the Ramachandran map. The calculated structures, which show that the N-terminal domain forms a homo-dimer in solution, is of high stereochemical quality as judged by the Ramachandran statistics (70% of the residues have backbone dihedral angles in the allowed region, 25.5% in the additionally allowed region, 4.0% in generously allowed region, and only 0.5% in disallowed region). Dynamics of nPS, which has rotational correlation time τc of 17.3 ns, was investigated by 15N relaxometry measurements. Results of these studies indicate that the E. coli protein exhibits dynamic nature at the dimer interface. These structural and dynamic features of the protein were found to be of interest when correlated with NMR based substrate binding studies.
Interaction of homo-dimeric amino-terminal domain (nPS) of E. coli pantothenate synthetase (PS; encoded by the gene panC; E.C. 6.3.2.1) with the substrates pantoate, β-alanine, ATP and the product pantothenate has been studied using isotopically edited solution NMR methods. Addition of pantoate prior to ATP has led to the interesting observation that pantoate binds each monomer of nPS at two sites. ATP displaces a molecule of pantoate from the ATP binding site. β-alanine and pantothenate do not bind the protein under the condition studied. Binding of pantoate and ATP also manifests as changes in residual dipolar couplings (RDCs) of backbone 1H-15N pairs in nPS when compared to the free form of the protein. Structures of homo-dimeric nPS bound to two molecules of pantoate (PDB entry 2k6e) as well as to pantoate + ATP (PDB entry 2k6f) were calculated by inclusion of hydrogen bonds between the ligands and backbone 1H-15N pairs of nPS in addition to other NMR derived restraints. The ligand bound structures have been compared to the similar forms of the M. tb PS. Structure of each monomer of nPS bound to pantoate and ATP shows the substrates in a favorable orientation for the intermediate pantoyl adenylate to form. Moreover, at all stages of substrate binding the symmetry of the dimer was preserved. A single set of resonances was observed for all protein-ligand complexes implying symmetric binding with full-occupancy of the ligands bound to the protein.
In an effort to understand the structural basis of the observed enzymatic properties of plant PS’s, a structural model of the Arabidopsis PS was constructed. The results of these structural and substrate binding studies strongly suggest that
1 Substrate binding to PS occurs only at the active site.
2 There are no additional substrate binding sites which could potentially participate as regulatory sites.
3 Pantoate does not bind at the dimer interface to induce the observed homotropic effects.
4 The structural results presented on the substrate bound forms of nPS have direct implication for the development of novel antibacterial and herbicidal agents.
Recently a great deal of interest has been evinced on the effects of molecular crowding on protein folding / unfolding pathways. Nuclear magnetic resonance is the only method by which high resolution structural information can be obtained on partially denatured states of a protein under equilibrium condition. Recent methodological advances have enabled the determination of high resolution structures using information embedded in the residual dipolar couplings.
Molecular crowding using confinement may thus reveal important details about chaperone mediated protein folding. We have attempted to develop a protocol to study the effects of molecular confinement by sequestering proteins in poly-acrylamide gels and then subjecting these protein molecules to denaturation and then characterize these states by nuclear magnetic resonance. The preliminary results of these studies are described here.
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Hendry, Garth S. "Dependence of substrate-water binding on protein and inorganic cofactors of photosystem II." Phd thesis, 2002. http://hdl.handle.net/1885/47151.
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The photosynthetic water oxidation reaction is catalyzed by an inorganic Mn4OxCaClyHCO3-z cluster at the heart of the oxygen evolving complex (OEC) in photosystem II. In the absence of an atomic resolution crystal structure, the precise molecular organization of the OEC remains unresolved. Accordingly, the role of the protein and inorganic cofactors of PSII (Ca2+, HCO3- and Cl-) in the mechanism of O2-evolution await clarification. In this study, rapid 18O-isotope exchange measurements were applied to monitor the substrate-water binding kinetics as a function of the intermediate S-states of the catalytic site (i.e. S3, S2 and S1) in Triton X-100 solubilized membrane preparations that are enriched in photosystem II activity and are routinely used to evaluate cofactor requirements. Consistent with the previous determinations of the 18O exchange behavior in thylakoids, the initial 18O exchange measurements of native PSII membranes at m/e = 34 (which is sensitive to the 16O18O product) show that the ‘fast’ and ‘slowly’ exchanging substrate-waters are bound to the catalytic site in the S3 state, immediately prior to O2 release. Although the slowly exchanging water is bound throughout the entire S-state cycle, the kinetics of the fast exchanging water remains too fast in the S2, S1 [and S0] states to be resolved using the current instrumentation, and left open the possibility that the second substrate-water only binds to the active site after the formation of the S3 state. Presented is the first direct evidence to show that fast exchanging water is already bound to the OEC in the S2 state. Rapid 18O-isotope exchange measurements for Ex-depleted PSII (depleted of the 17- and 23-kDa extrinsic proteins) in the S2 state reveals a resolvable fast kinetic component of 34k2 = 120 ± 14 s-1. The slowing down of the fast phase kinetics is discussed in terms of increased water permeation and the effect on the local dielectric following removal of the extrinsic subunits. In addition, the first direct evidence to show the involvement of calcium in substrate-water binding is also presented. Strontium replacement of the OEC Ca2+-site reveals a factor of ~3-4 increase in the 18O exchange of the slowly exchanging water across the S3, S2 and S1 states while the kinetics of the fast exchanging water remain unchanged. Finally, a re-investigation of the proposed role for bicarbonate as an oxidizable electron donor to photosystem II was unable to discern any 18O enrichment of the photosynthetically evolved O2 in the presence of 18O-bicarbonate. A working model for O2-evolution in terms of these results is presented.
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Wisniewska, Magdalena [Verfasser]. "Biochemical studies on IGF and IGF-binding proteins interactions & structural investigations on the SH3 domain of Crk-associated tyrosine kinase substrate p130cas (CAS) / Magdalena Wisniewska." 2005. http://d-nb.info/978198549/34.
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Mühlhausen, Helene. "Untersuchungen zur molekularen Ursache der Multiplen Sulfatase-Defizienz: Reinigung, Funktions- und Strukturanalyse von varianten Proteinen des Formylglycin-generierenden Enzyms." Doctoral thesis, 2014. http://hdl.handle.net/11858/00-1735-0000-0023-9953-2.
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Pinto, Bárbara Palma de Abreu Caldeira. "Studies on the function and substrate permeation in ABC transporters by biomolecular simulations." Doctoral thesis, 2021. http://hdl.handle.net/10362/121941.
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"The translocation of molecules across cell membranes often requires the participation of transmembrane proteins. The ATP Binding Cassette (ABC) transporters are one of the major class of proteins dedicated to the translocation of molecules across membranes. ABC transporters make use of ATP hydrolysis and undergo a series of large-scale conformational changes in order to carry out their function. Despite being extensively studied, some molecular details regarding their function remain undisclosed. In this thesis, the function of three relevant ABC systems, CFTR, MsbA and the Escherichia coli MalFGK2E importer, was investigated using computational methods, such as molecular dynamics simulations. The effect of the CFTR mutation F508del, responsible for cystic fibrosis, was studied in the nucleotide binding domains of the CFTR receptor. In the MsbA protein, the molecular details of nucleotide binding during the adenylate kinase cycle were investigated in collaboration with experimental groups with expertise in solid state NMR and EPR. Finally, the details of substrate translocation in the MalFGK2E importer were also researched.(...)"
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Fisher, Loren Tichauer. "Role of a topologically conserved Isoleucine in the structure and function of Glutathione Transferases." Thesis, 2006. http://hdl.handle.net/10539/1726.
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Student Number : 0002482E -
MSc dissertation -
School of Molecular and Cell Biology -
Faculty of ScienceProteins in the glutathione transferase family share a common fold. The close packing of secondary structures in the thioredoxin fold in domain 1 forms a compact hydrophobic core. This fold has a bababba topology and most proteins/domains with this fold have a topologically conserved isoleucine residue at the N-terminus of a-helix 3. Class Alpha glutathione transferases are one of 12 classes within the glutathione transferase family. To investigate the role of the conserved isoleucine residue in the structure, function and stability of glutathione transferases, homodimeric human glutathione transferase A1-1 (hGST A1-1) was used as a representative of the GST family. Ile71 was replaced with valine and the properties of I71V hGST A1-1 were compared with those of wildtype hGST A1-1. The spectral properties monitored using far-UV CD and tryptophan fluorescence indicated little change in secondary or tertiary structure confirming the absence of any gross structural changes in hGST A1-1 due to the incorporation of the mutation. Both wildtype and mutant dimeric proteins were determined to have a monomeric molecular mass of 26 kDa. The specific activity of I71V hGST A1-1 (130 mmol/min/mg) was three times that of wildtype hGST A1-1 (48 mmol/min/mg). I71V hGST A1-1 showed increased kinetic parameters compared to wildtype with a 10-fold increase in kcat/Km for CDNB. The increase in Km of I71V hGST A1-1 suggests the mutation had a negative effect on substrate binding. The DDG for transition state stabilisation was –5.82 kJ/mol which suggest the I71V mutation helps stabilise the transition state of the SNAR reaction involving the conjugation of reduced glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB). A 2-fold increase in the IC50 value for I71V hGST A1-1 (11.3 mM) compared to wildtype (5.4 mM) suggests that the most noticeable change due to the mutation occurs at the H-site of the active site. Conformational stability studies were performed to determine the contribution of Ile71 to protein stability. The non-superimposability of I71V hGST A1-1 unfolding curves and the decreased m-value suggest the formation of an intermediate state. The conformational stability of I71V hGST A1-1 (16.5 kcal/mol) was reduced when compared to that of the wildtype (23 kcal/mol). ITC was used to dissect the binding energetics of Shexylglutathione to wildtype and I71V hGSTA1-1. The ligand binds 5-fold more tightly to wildtype hGST A1-1 (0.07 mM) than I71V hGST A1-1 (0.37 mM). The I71V mutant displays a larger negative DCp than wildtype hGST A1-1 (DDCp = -0.41 kJ/mol/K). This indicates that a larger solvent-exposed hydrophobic surface area is buried for I71V hGST A1-1 than for wildtype hGST A1-1 upon the binding of S-hexylglutathione. Overall the results suggest that Ile71 conservation is for the stability of the protein as well as playing a pivotal indirect role in catalysis and substrate binding.
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Wagner, Judith Nastjenka [Verfasser]. "The HSP100 protein ClpA : significance of the N-terminal domain in substrate recognition and binding and characterization of the novel substrate RepE / vorgelegt von Judith Nastjenka Wagner." 2008. http://d-nb.info/991293142/34.
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Barker, Megan. "Structural Investigation of Processing α-Glucosidase I from Saccharomyces cerevisiae." Thesis, 2010. http://hdl.handle.net/1807/32660.
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N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications.
The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work.
To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs.