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1
Lundell, Sandra J. "Quantum Mechanical Studies of N-H···N Hydrogen Bonding in Acetamide Derivatives and Amino Acids." DigitalCommons@USU, 2018. https://digitalcommons.usu.edu/etd/7309.
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Proteins are made of vast chains of amino acids that twist and fold into intricate designs. These structures are held in place by networks of noncovalent interactions. One of these, the hydrogen bond, forms bridges between adjacent pieces of the protein chain and is one of the most important contributors to the shape and stability of proteins. Hydrogen bonds come in all shapes and sizes and a full understanding of these not only aids in our understanding of proteins in general but can bridge the gap to finding cures to many protein-related diseases, such as sickle-cell anemia. The primary aim of this thesis is to discover if a specific type of hydrogen bond, the N-H···N bond, occurs within proteins and if so, if it contributes to the structure and stability of proteins.
2
Huffman, Jennifer Elizabeth. "Genetic analysis of protein N-glycosylation." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/10038.
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The majority of human proteins are post-translationally modified by covalent addition of one or more complex oligosaccharides (glycans). Alterations in glycosylation processing are associated with numerous diseases and glycans are attracting increasing attention both as disease biomarkers and as targets for novel therapeutic approaches. Using a recently developed high performance liquid chromatography (HPLC) method for high-throughput glycan analysis, genome-wide association studies (GWAS) of 33 directly measured and 13 derived N-glycan features were performed in 3533 individuals from four European isolated populations. Polymorphisms at six loci were found to show genome-wide significant association with plasma concentrations of N-glycans. Several of these gene products have well characterised roles in glycosylation, however, SLC9A9 and HNF1A were two of the novel findings. Subsequent work performed by collaborators found HNF1A to be a “master regulator” of genes involved in the fucosylation of plasma N-glycans. Additionally, this work led to the discovery that N-glycans could act as biomarkers to discriminate HNF1A-MODY from type 1 and type 2 diabetes mellitus (T1D, T2D) patients. After the success of the total plasma N-glycan GWAS, it was thought that stronger and more biologically interpretable associations may be found from the investigation of N-glycans isolated from a single protein. Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic networks that govern IgG glycosylation, N-linked IgG glycans were quantitated using ultra performance liquid chromatography (UPLC) in 2247 individuals from the same four European populations from the previous study. GWAS of the 77 N-glycan measures identified 15 loci with a p-value < 5x10-08. Four loci contained genes encoding glycosyltransferases, while the remaining loci contained genes that have not previously been implicated in protein glycosylation. However, most have been associated with autoimmune and inflammatory conditions and/or hematological cancers. Several high-throughput methods for the analysis of N-glycans have been developed in the past few years but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. To this end, four of these methods were compared, UPLC, multiplexed capillary gel electrophoresis (xCGE), and two mass spectrometric (MS) methods, for quantitative profiling of N-glycosylation of plasma IgG in a subset of 1201 individuals recruited from two of the cohorts used in the previous GWAS studies. A “minimal” dataset was compiled of N-glycan structures able to be measured by all four methods. To evaluate their accuracy, correlations were calculated for each structure in the minimal dataset. Additionally, GWAS was performed to test if the same associations would be observed across methodologies. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and xCGE, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should aid in the selection of the most appropriate method for future studies. This work shows that it is possible to identify new loci that control glycosylation of plasma proteins using GWAS and the potential of N-glycans for biomarker development. It also provides some guidelines for methodology selection for future studies of N-glycans.
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Agah, Sayeh. "Parvalbumin stability and calcium affinity : the impact of the n-terminal domain /." Free to MU Campus, others may purchase, 2004. http://wwwlib.umi.com/cr/mo/fullcit?3164486.
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Lucas, Olivier. "Molecular and systemic functions of the vertebrate-specific TATA-binding protein N terminus." Diss., Montana State University, 2009. http://etd.lib.montana.edu/etd/2009/lucas/LucasO0509.pdf.
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Woollard, Geoffrey Robert Paget. "Redesign of the N-end rule protein ClpS for use in high-throughput N-end protein sequencing." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/46377.
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Current protein sequencing methods include mass spectrometry and Edman degradation. We envision a novel high-throughput protein sequencing method using affinity adapters to recognize the N-terminal residue of a denatured peptide in an iterative process. This thesis takes a first step toward designing robust and selective affinity reagents. We outline our pipeline for designing selective protein adapters that recognize the N-terminal amino acid of a peptide independent of the following sequence. We based our design on a substrate recognition protein in the N-end rule pathway, ClpS. The bacterial N-recognin protein ClpS binds peptide substrates, termed N-degrons, that have a bulky hydrophobic amino acid (L/F/Y/W) at the N-terminus. Using full atom in silico models we designed hydrogen bonding and salt-bridge contacts in ClpS to novel N-degron substrates (N-end D/E/T), predicted the selectivity of these designs, and experimentally verified them. Of 11 designs, we purified nine that were soluble by SDS-PAGE, and obtained a peptide binding profile to 30 peptides with a modified ELISA assay. Most designs were non-specific or had no binding affinity. Four designs M53A, L112F, I45L, I45L_I45L_M53A had an increase in affinity to various substrates, but were not selective as they retained affinity to the native substrates (N-end L/F/Y/W). We performed molecular dynamics simulations on several proteins that were soluble or insoluble under standard expression conditions in E. coli, in order to learn parameters that were indicative of kinetic instability. Using a back-to-consensus approach, we identified a point mutant S104F that stabilizes the scaffold of ClpS as assayed by GFP fluorescence in a GFP-ClpS fusion protein. This thesis outlines the computational design pipeline we developed, which includes a RosettaScripts protocol, an in silico selectivity screen with AutoDock, and a kinetic stability confidence score from a molecular dynamics trajectory. Finally, we make suggestions toward designing selective affinity reagents for high-throughput N-end protein sequencing.
6
Wood, Alison. "N-linked protein glycosylation in Helicobacter species." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/nlinked-protein-glycosylation-in-helicobacter-species(ef97ffdd-aca4-40ff-b52b-7b8ca030bc82).html.
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N-linked protein glycosylation involves the transfer of a glycan onto an Asparagine residue (N) of a polypeptide chain. It is common in Eukaryotes and has recently been observed in Prokaryotes, most notably in Campylobacter jejuni. The C. jejuni N-linked glycosylation system is encoded on a single pgl gene locus that also functions when expressed in Escherichia coli. The key enzyme involved in N-linked protein glycosylation is encoded by the pglB gene and transfers lipid-linked glycan onto N residues of glycoproteins in the periplasm. It is clear from accumulating genome sequence data that pglB orthologues are present in all Campylobacter species and in related species such as Wolinella succinogenes, Desulfovibrio vulgaris and Desulfovibrio desulfuricans. Most Helicobacter species, including Helicobacter pylori, lack the pglB gene but three related Helicobacter species Helicobacter pullorum, Helicobacter canadensis and Helicobacter winghamensis have two distinct pglB genes. These and other orthologues of C. jejuni pgl genes are located not within a single locus but rather at five distinct loci. One of the two pglB genes, termed pglB1, is required for in vitro N-glycosylation of peptides (Jervis et al., 2010). In this thesis I present data on the role of further pgl gene orthologues and previously uncharacterized genes in H. pullorum N-glycosylation. Furthermore I have also identified a number of H. pullorum glycoproteins and provide data comparing N-glycosylation processes in C. jejuni and H. pullorum. These data expand our preliminary observations on the first Helicobacter N-linked glycosylation system, and provide important information on the similarities and differences between the well characterised C. jejuni system and these more recently identified pathways.
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Karlsson, Gunilla Birgitta. "Effects of the imino sugar N-butyldeoxynojirimycin on protein N-linked glycosylation." Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.333246.
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Raggett, Elaine. "The structure and function of translocation domain of Colicin N." Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285400.
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Murray, Anne Riché. "The functional significance of rhodopsin's N-linked glycosylation." Oklahoma City : [s.n.], 2009.
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Su, Wei. "Characterization of N-GAIP, a novel RGS protein." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0010/MQ40774.pdf.
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Zamiri, Maryam. "Synthesis of protein arginine N-methyltransferase 6 inhibitors." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43808.
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Protein arginine N-methyltransferases (PRMTs) are pertinent targets for drug discovery as their dysfunction is associated with a number of diseases such as cancers, cardiovascular diseases and viral pathogenesis. The precise role of PRMTs in the initiation, development, or progression of diseases is not known yet.
Due to association of PRMT1 and 4 with transcriptional activation, the main focus of inhibitor discovery has been on these two enzymes. On the other hand, the goal of this study is to find a PRMT6 specific inhibitor. PRMT6 methylates DNA polymerase β, histones H3 and H4 and HIV proteins: Rev and Tat.
PRMT6 uses S-adenosyl-L-methionine (AdoMet) as the “methyl group” source. AdoMet fits into a distinct conserved binding site in the enzyme, which is located adjacent to the protein substrate/catalytic site such that its S⁺-Me motif is correctly positioned with respect to the substrate arginine nitrogen atom that undergoes methylation.
Based on crystallography data for PRMT1, the purine C8 center in AdoMet is in close proximity to the methionine sulfur atom (M166 in PRMT6). As shown by Frankel et al. (Faculty of Pharmaceutical Sciences, UBC), the M166C PRMT6 mutant displays activity. Based upon this observation, we hypothesize that Ado-Met analogues with reactive substituents (e.g., CHO) at C8 position of adenine ring will form a covalent bond with the proximal Cys SH group in M166C PRMT6. This validates our further hypothesize that in appropriately designed analogues, it will be possible to subsequently detach the sugar and amino acid components of Ado-Met to leave the adenine ring component alone bound to the enzyme. This provides a unique opportunity to explore the “fragment based approach in drug discovery” to design PRMT6 specific inhibitors.
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Mackie, Brianna D. "Chemical Probes for Protein α-N-Terminal Methylation." VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/4880.
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While protein α-N-terminal methylation has been known for nearly four decades since it was first uncovered on bacteria ribosomal proteins L33, the function of this modification is still not entirely understood. Recent discoveries have demonstrated α-N-terminal methylation is essential to stabilize the interactions between regulator of chromosome condensation 1 (RCC1) and chromatin during mitosis, to localize and enhance the interaction of centromere proteins (CENPs) with chromatin, and to facilitate the recruitment of DNA damage-binding protein 2 (DDB2) to DNA damage foci. Identification of N-terminal methyltransferase 1 (NTMT1) unveiled the eukaryotic methylation writer for protein α-N-termini. In addition, NTMT2 that shares over 50% sequence similarity, has been identified as another mammalian protein α-N-terminal methylation writer. Knockdown of NTMT1 results in mitotic defects and sensitizes chemotherapeutic agents in breast cancer cell lines, while NTMT1 knockout mice showed premature aging. Additionally, NTMT1 has been shown to be overexpressed in a colorectal and melanoma tumor tissues, and in lung and liver cancer cell lines.
Given the vast array of clinical relevance, chemical probes and inhibitors for NTMT1 are vital to elucidate information about the function and downstream process of protein α-N-terminal methylation. Therefore, 47 peptidomimetic compounds have been synthesized that target NTMT1. These peptide-based compounds range from three to six amino acids in length and the top 5 compounds have 3- to 300- fold selectivity for NTMT1 compared to other methyltransferases. An inhibition mechanism study has also been performed to verify the inhibitors are targeting the NTMT1 peptide binding site. Seven compounds have an IC50 of less than 5 µM and our top inhibitor, BM-47, has an IC50 of 0.32 µM ± 0.06 for NTMT1.
To further elucidate information about the NTMTs and their downstream effects, we utilized photoaffinity probes to target these enzymes. Our 6 photoaffinity probes exhibited in a dose- and time-dependent manner. Probe labeling has been shown to be driven by recognition and selectively and competitively label the NTMT writers in a complex cellular mixture. Our results also provided the first indication of substrate preferences among NTMT1/2. Methylated photoaffinity probes were also synthesized to identify novel proteins that recognize a methylated N-terminus and shed light on the function of α-N-terminal methylation.
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Eifan, Saleh A. "Functional analysis of the orthobunyavirus nucleocapsid (N) protein." Thesis, St Andrews, 2008. http://hdl.handle.net/10023/542.
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Glover, William Broc. "N-beta-methylamino-L-alanine : a non-protein amino acid incorporated into protein." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/50730.
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N-β-methylamino-L-alanine (BMAA) is a naturally-occurring toxin produced in cyanobacteria that has been linked to neurological degeneration. Efforts to study and quantify BMAA in the environment are hampered by two biologically-occurring isomers, N-(2- aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB), that exist in very low concentrations in many sample matrices, indicating a need for improved analytical methods.
The first objective of this thesis is to develop an accurate, precise and sensitive method for the analysis of BMAA and the isomers AEG and DAB that is applicable to a wide variety of sample matrices. A method for complete chromatographic separation of the isomers was developed using chemical derivatization, reversed phase chromatography and tandem mass spectrometry, and was validated with cyanobacteria-containing natural health products.
The mechanism of toxicity of BMAA is not fully understood, and previous studies have shown an association between BMAA and protein. The second objective of this thesis is to determine whether BMAA is incorporated into protein via a modified proteomics approach used to identify peptides containing BMAA in tissue from post-mortem human patients. This approach identified BMAA in proteins isolated from the frontal lobe of Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease (AD), and control patients. To determine whether BMAA incorporation into protein occurs as an error in synthesis, a cell-free expression system was challenged with a deficiency of specific amino acids and supplemented with BMAA. BMAA was incorporated into protein via an error in synthesis. Together, these data demonstrate accurate methods for quantification of BMAA, a route of exposure through dietary products, and a potential mechanism for storage of BMAA in proteins.Irving K. Barber School of Arts and Sciences (Okanagan)
Biology, Department of (Okanagan)
Graduate
15
Sealey, Amy Lynn. "Loss of the murine TATA-binding protein N terminus leads to placental labyrinth defects but not maternal adaptive immune responses." Thesis, Montana State University, 2007. http://etd.lib.montana.edu/etd/2007/sealey/SealeyA0507.pdf.
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Koch, Kerstin. "Statistical analysis of amino acid side chain flexibility for 1:n protein protein docking." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=968919413.
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Jiménez, Flores Lizbeth Minerva. "The Role of Protein Kinase N in Gastric Cancer." Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/664078.
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Gastric cancer (GC) is one of the most common types of cancer in the Western World and accounts for over 700,000 deaths every year worldwide. The prognosis is dismal, with an average 5-year survival rate of less than 20%, mainly because of late diagnosis, due to the early stages are clinically silent. The cause of GC is multifactorial, as infectious agents, enviromental or/and genetic factors. Based on Lauren’s histologic classification, there are 2 types of GC: intestinal (IGC) and diffuse (DGC). Diffuse carcinoma cells lacks cohesion and invade tissues independently or in small clusters, is more common in young patients and behaves more aggressively than the intestinal type. Recent findings published in Nature and Nature Genetics identified frequent hotspots (14-24%) mutations in the small GTPase RHOA in the diffuse type of gastric tumors. To investigate the mechanism that underlies downstream of RHOA we analyzed the capacity of the mutations more commonly found in gastric tumors to bind to different effectors. We observed that the mutations found in gastric tumors specifically affect the capacity of RHOA to bind to PKN effector family. Therefore, in this thesis we study and characterized the role of PKN1 in GC. We demonstrated that the downregulation of PKN1 with shRNA or the deletion mediated by CRISPR/Cas9 results in the increase of proliferation in the diffuse gastric cell lines “in vitro” and “in vivo”. Moreover, the opposite effect is observed when we overexpress the constitutively active form of PKN1 in diffuse gastric cell lines with moderate or low levels of PKN1. In addition, we use a novel mouse model with conditional expression in the gastric mucosa of the RHOA-Y42C mutation (the most frequent mutation found in DGC) to investigate the role of RHOA in gastric tumorogenesis initiated by either N-methyl-N-nitrosourea (MNU) or Apc mutations. The mice with expression of the RHOA- Y42C have a significant increase in the number of tumors indicating that RhoA-Y42C is important for progression of the gastric tumors. Finally, we performed a preclinical testing of a new therapeutic approach, such as dietary supplements of arachidonic acid, a well- established activator of PKN1. The work carried out in this thesis will shed light on the newly identified deregulation of RHOA-PKN signaling in gastric cancer and provide a solid rationale for therapeutic targeting of this pathway.
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Lexis, Meike [Verfasser], and N. [Akademischer Betreuer] Willenbacher. "Rheology of Protein Foams / Meike Lexis. Betreuer: N. Willenbacher." Karlsruhe : KIT-Bibliothek, 2015. http://d-nb.info/1073939871/34.
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Pak, Laam. "Insights into a heteromeric protein arginine N-methyltransferase complex." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/42123.
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Protein arginine N-methyltransferases (PRMTs) act in signaling pathways and gene
expression by methylating arginine residues within target proteins. PRMT1 is responsible for
most cellular arginine methylation activity and can work independently or in collaboration with
other PRMTs. In this Ph.D. thesis I demonstrated an interaction between PRMT1 and -2 using
co-immunoprecipitation and bimolecular fluorescence complementation (BiFC). As a result of
this interaction, PRMT2 stimulated PRMT1 methyltransferase activity, affecting its apparent
Vmax and Km values in vitro, and increasing the production of methylarginines in cells. Active
site mutations and regional deletions on PRMT1 and -2 were also investigated, which
demonstrated that complex formation required full-length, active PRMT1. However, the
interaction between PRMT1 and -2 proved insensitive to methylation inhibition in the absence of
the PRMT2 Src homology 3 (SH3) domain, which suggests that the PRMT2 SH3 domain may
mediate this interaction between PRMT1 and -2 in a methylation-dependent fashion.
The role of the PRMT2 SH3 domain was investigated through screening for its associated
proteins using GST-pull down assays followed by LC-MS/MS proteomic analysis. The result of
this study revealed associations of the PRMT2 SH3 domain with at least 29 splicing-related
proteins, suggesting a potential role of PRMT2 in regulating pre-mRNA processing and splicing.
The interaction between PRMT2 and the Src substrate associated in mitosis of 68 kDa (Sam68)
possibly through the PRMT2 SH3 domain was demonstrated using co-immunoprecipitation.
Additionally, immunofluorescence results present herein imply that the PRMT2 SH3 domain
could affect Sam68 sub-cellular localization in hypomethylated HeLa cells. The biological functions of PRMT2 and the PRMT1/2 heteromeric complex were explored
by pursuing the identity of associated proteins common to both PRMT1 and -2 using mass
spectrometry proteomics. Approximately 50% of the identified protein hits have reported roles
in controlling gene expression, while other hits are involved in diverse cellular processes such as
protein folding, degradation, and metabolism. Importantly, three novel PRMT2 binders, p53,
promyelocytic leukemia protein (PML), and extra eleven nineteen (EEN) were uncovered,
suggesting that PRMT2 could participate in regulation of transcription and apoptosis through
PRMT2-protein interactions.
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Ntwasa, Monde McMillan. "The studies of Drosophila Myristoyl-CoA : protein N-myristoyltransferase." Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627451.
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Ikenaka, Yasuhiro. "Protein Engineering of N-Carbamyl-D-Amino Acid Amidohydrolase." Kyoto University, 1999. http://hdl.handle.net/2433/181917.
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Frost, Helen. "N-linked protein glycosylation in Campylobacter and Helicobacter species." Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/nlinked-protein-glycosylation-in-campylobacter-and-helicobacter-species(ca49728c-1406-463f-bead-99d7cf336cb9).html.
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N-linked protein glycosylation is the enzymatic transfer of a carbohydrate glycan to an asparagine residue of a polypeptide, catalysed by an N-oligosaccharyl transferase(OTase). Bacterial N-glycosylation is best understood in the foodborne pathogen Campylobacter jejuni, in which a heptasaccharide glycan is built at cytoplasmic face of the inner membrane, flipped to the periplasm and transferred to a polypeptide enbloc. C. jejuni encodes each of the proteins required for the N-glycosylation pathway in a single genetic region, termed the pgl locus. Homologues of the gene encoding the C. jejuni OTase, PglB, are found in all Campylobacter species, three Helicobacter species, and more distantly related ε- and δ-Proteobacteria species such as Wolinella succinogenes, Desulfovibrio desulfuricans and Nitratiruptor tergarcus. A small numberof Campylobacter species and all three pglB-containing Helicobacter species have two distinct pglB genes, pglB1 and pglB2, along with homologues of other C. jejuni pgl genes. The work presented in this thesis investigated the N-glycosylation system of a bacterial species encoding two distinct PglBs, C. concisus. The roles of the two PglB enzymes in C. concisus were investigated using an in vitro OTase assay, and the structure of a C. concisus N-glycan elucidated by mass spectrometry. The work in this thesis also expands our knowledge of C. jejuni N-glycosylation by investigating the full scope of N-glycosylation using an in silico method to predict the total C. jejuni N-glycoproteome. This was followed by experimental validation of these predictions, in which three novel C. jejuni N-glycoproteins were identified, bringing the number of reported C. jejuni NCTC 11168 N-glycoproteins to 57. One of these novel N-glycoproteins, Cj0633, is the most extensively N-glycosylated bacterial glycoprotein reported to date, with the addition of up to eight N-glycans when expressed in the presence of the C. jejuni pgl machinery. In the final investigation presented in this thesis, a method to identify bacterial N-glycoproteins using anti-glycan antisera to immunoprecipitate N-glycoproteins was developed. These data expand our knowledge of C. concisus N-glycosylation and provide valuable insight into the full scope of N-glycosylation in C. jejuni.
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Liu, Yuxin. "Pctaire1 phosphorylates N-ethylmaleimide sensitive fusion protein and regulates exocytosis /." View abstract or full-text, 2006. http://library.ust.hk/cgi/db/thesis.pl?BICH%202006%20LIU.
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Fatkins, David G. "N(EPSILON)-THIOACETYL-LYSINE AS A MULTIFACETED TOOL FOR ENZYMATIC PROTEIN LYSINE N(EPSILON)-DEACETYLATION." University of Akron / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=akron1185377018.
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Kam, Yuet Fong. "Regulation of c-jun n-terminal kinases by opioid receptors /." View Abstract or Full-Text, 2002. http://library.ust.hk/cgi/db/thesis.pl?BICH%202002%20KAM.
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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002.Includes bibliographical references (leaves 83-103). Also available in electronic version. Access restricted to campus users.
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Wright, Megan Holly. "Chemical tools for probing protein N-myristoylation in protozoan parasites." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/39376.
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Protein N -myristoylation is the attachment of a 14-carbon fatty acid, myristate, onto the N-terminal residue of specific proteins. This co - and post-translational modification is catalysed by myristoyl CoA:protein N -myristoyltransferase (NMT), an essential enzyme in eukaryotes. N -Myristoylated proteins have diverse roles, but typically localise to cellular membranes where many are thought to be involved in signalling or trafficking processes. NMT is a potential therapeutic target in diseases caused by parasitic protozoa, such as malaria, leishmaniasis and African sleeping sickness. N -Myristoylation is difficult to study by conventional biochemical methods. Recently chemical proteomics has emerged as a powerful technique for probing protein lipidation. In this approach chemically tagged myristic acid substrate analogues are incorporated metabolically into lipidated pro teins. Tagged proteins can then be selectively 'captured' after cell lysis via a bioorthogonal ligation reaction to install a variety of labels for further analysis. This thesis describes the development of such an approach for profiling protein lipidation in diverse systems, including human cells, Plasmodium , Leishmania and T. brucei parasites. This work includes the the synthesis of multifunctional probes for the capture of tagged proteins and characterisation of a tagged myristate analogue as a Plasmodium NMT substrate. These tools are applied in several contexts, with analysis by a combination of gel-based methods and mass spectrometry. The first de novo identification of N -myristoylated proteins in P. falciparum , T. brucei and L. donovani is described. The chemical proteomic methodology developed is also used to profile lipidation in the presence of putative NMT inhibitors to assess whether compounds act on -target in live parasites. The correlation between efficacy against the cell and on-target activity is analysed to explore NMT as a potential drug target in malaria and leishmaniasis.
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Brooke, Edward W. "Protein-ligand interactions of arylamine N-acetyltransferase from Mycobacterium smegmatis." Thesis, University of Oxford, 2003. http://ora.ox.ac.uk/objects/uuid:f13c3191-b098-4a85-84c3-90590b365d30.
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Tuberculosis is the world's largest cause of death from an infectious agent. Treatment is by an extended period of combination chemotherapy. Drug resistance is an increasing problem in tuberculosis therapy, particularly to the frontline anti-tubercular drug isoniazid (INH). Recombinant arylamine N-acetyltransferase (NAT) of Mycobacterium tuberculosis N-acetylates INH using the cofactor Acetyl Coenzyme A. NAT from M. tuberculosis is a polymorphic enzyme and also acetylates INH in vivo. Acetylated INH is inactive therapeutically against M. tuberculosis both in vivo and in vitro. The acetylation of isoniazid in the mycobacterial cell may compete with the activation of INH by the catalase-peroxidase, katG, and hence contribute to INH resistance in clinical isolates. Inhibition of NAT in M. tuberculosis may thus increase the efficacy of INH therapy. A novel assay based around the detection of free Coenzyme A released during the acetylation reaction was used to determine the substrate specificity of recombinant NAT from the related Mycobacterium M. smegmatis (MSNAT). A relationship was observed between the lipophilicity of simple arylamine substrates and the rate of acetylation by MSNAT. Several MSNAT substrates possess antibacterial activity. The assay could also be used to screen compound libraries for MSNAT inhibitors. Synthesis of seventeen thiazolidinedione sultams in collaboration with Dr.Vickers (Dyson Perrins), identified as weak inhibitors of MSNAT, gave a minimum competitive inhibitory constant of 14μM. Screening a library of 5,074 drug-like compounds for inhibition of MSNAT identified thirteen compounds with semi-maximal inhibition constants (IC50) of below 10μM. Based on this, fifteen maleimides were synthesised and were irreversible inhibitors of MSNAT with submicromolar potency. Similarly, ninety-six aminothiazoles were synthesised by Dr. Vickers and were uncompetitive inhibitors of MSNAT with a minimum IC50 of 1.5μM. The most potent aminothiazole showed no effect on the growth of M. smegmatis or M. bovis BCG or the sensitivity of the bacteria to isoniazid. However the aminothiazoles were shown not to penetrate the cells.
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Anthony, Shelagh. "Analysis of mammalian protein arginine N-methyltransferases in the vasculature." Thesis, University College London (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424909.
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Stroukova, Daria. "Recombinant expression and characterisation of the colicin N immunity protein." Thesis, University of Newcastle upon Tyne, 2016. http://hdl.handle.net/10443/3250.
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Some Escherichia coli express plasmid encoded toxins called colicins to eliminate ecological competitors. Colicins are a diverse group of toxins, usually divided into three groups: nucleases, pore-formers and peptidoglycan synthesis inhibitors. In addition, each colicinogenic plasmid codes for an immunity protein - a self-protection mechanism against its own toxin. Immunity proteins are highly specific to their cognate toxins but it is not clearly known how immunity proteins to pore-forming colicins achieve protection. This work focuses on the immunity protein for the smallest pore-forming colicin, colicin N. To investigate in vitro how the Colicin N immunity protein (CNI) neutralises the toxin, different overexpression and purification methods were tested. The fusion CNI-3C-HALO7-6His yielded the most protein, when expressed in C41 E. coli cells at 37 °C overnight in Terrific Broth. The fusion protein increases resistance to Colicin N dramatically and is therefore folded and localised correctly. Decyl β-Dmaltopyranoside is the most effective detergent to solubilise and stabilise the protein fusion. To investigate if CNI inactivates ColN by binding to it via hydrophobic α-helical interactions, the protein’s helical regions were defined in silico and swapped with the respective parts of the Colicin A immunity protein, CNI’s closest homologue, which provides immunity to Colicin A. The N-terminal region of CNI, including the first transmembrane helix, is not necessary for specific CNI-Colicin N interaction but the other helices are crucial for protein stability and function. CNI can be expressed in an active form fused to GFP for microscopy. Epifluorescent and TIRF microscopy revealed that CNI is localised to the cell periphery and appears to be very evenly distributed in the cell membrane. Colicin N addition does not affect CNI distribution. Single molecule tracking in TIRF microscopy showed that the diffusion rate of CNI does not change when ColN is added. Future work can pursue either of the three approaches used here to study CNI using in vitro structural analysis, site-directed mutagenesis of the active site or in vivo interaction of CNI with other E. coli inner membrane proteins.
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Caufield, J. Harry. "N-TERMINAL PROCESSING OF RIBOSOMAL PROTEIN L27 IN STAPHYLOCOCCUS AUREUS." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/361.
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The bacterial ribosome is essential to cell growth yet little is known about how its proteins attain their mature structures. Recent studies indicate that certain Staphlyococcus aureus bacteriophage protein sequences contain specific sites that may be cleaved by a non-bacteriophage enzyme (Poliakov et al. 2008). The phage cleavage site was found to bear sequence similarity to the N-terminus of S. aureus ribosomal protein L27. Previous studies in E. coli (Wower et al.1998; Maguire et al. 2005) found that L27 is situated adjacent to the ribosomal peptidyl transferase site, where it likely aids in new peptide formation. The predicted S. aureus L27 protein contains an additional N-terminal sequence not observed within the N-terminus of the otherwise similar E. coli L27; this sequence appears to be cleaved, indicating yet-unobserved ribosomal protein post-translational processing and use of host processes by phage. Phylogenetic analysis shows that L27 processing has the potential to be highly conserved. Further study of this phenomenon may aid antibiotic development.
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Ottman, Michael J., Michael D. Sheedy, and Richard W. Ward. "Late Season N Application Method Effect on Grain Protein, 2016." College of Agriculture, University of Arizona (Tucson, AZ), 2016. http://hdl.handle.net/10150/625425.
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4 pp.Nitrogen fertilizer is normally applied later in the season around flowering time to boost grain protein content. The purpose of this study is to determine if the grain protein boost provided by late N application is affected by method of application. A trial testing late season N application methods was conducted at the Maricopa Ag Center in the 2016 growing season. The crop was grown 211 lb N/acre in split applications until flowering when 35 lb N/acre was applied as UAN32 in the irrigation water (fertigation), as low biuret urea in a foliar application, or as urea granules compared to no N application at all at flowering. In this study, we were not able to detect a difference in grain protein or any other variable measured due to the late N application method. We did measure a 0.4% increase in grain protein regardless of late season N application method compared to the control with no late N applied.
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VANTARAKI, CHRISTINA. "Counteraction of urea-induced protein denaturation by Trimethylamine N-oxide." Thesis, Uppsala universitet, Molekyl- och kondenserade materiens fysik, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-390487.
Full textAbstract:
A common consequence of protein denaturation is the loss of biological activity. Natural osmolytes such as Trimethylamine N-oxide (TMAO) contribute to protein folding, whereas other osmolytes such as urea act as an agent in the denaturation of proteins. Many studies have shown that denaturation of proteins could occur for certain concentrations of urea, however, this effect could be prevented with the presence of Trimethylamine N-oxide (TMAO) molecules. The aim of the present study is to find out the mechanism of TMAO as a protein stabilizer against urea. Firstly, Molecular Dynamics simulations were carried out for 1, 8, 27 and 64 TMAO molecules. The time-average location of the TMAO molecules during the simulation was studied by the partial density. These simulations examine if TMAO is amphiphilic molecule, i.e contains both hydrophobic and hydrophilic parts. However, these results might not be representative due to bad statistics. Secondly, an experiment ran at BESSY II at Helmholtz-Zentrum Berlin using X-ray Photoelectron Spectroscopy in liquids. In this experiment, Lauryldimethylamine oxide(LDAO) was used instead of Trimethylamine N-oxide (TMAO) due to some practical reasons. The behaviour of urea and LDAO molecule was studied when these molecules were in different and same solutions. The purpose of this experiment is to find out the mechanism of LDAO against urea. Finally, LDAO interacts with urea and a possible mechanism between them is suggested. A common consequence of protein denaturation is the loss of biological activity. Natural osmolytes such as Trimethylamine N-oxide (TMAO) contribute to protein folding, whereas other osmolytes such as urea act as an agent in the denaturation of proteins. Many studies have shown that denaturation of proteins could occur for certain concentrations of urea, however, this effect could be prevented with the presence of the Trimethylamine N-oxide (TMAO) molecules. The aim of the present study is to find out the mechanism of TMAO as a protein stabilizer against urea. Firstly, Molecular Dynamics simulations were carried out for 1, 8, 27 and 64 TMAO molecules. The time-average location of TMAO molecules during the simulation was studied by the partial density. These simulations examine if TMAO is amphiphilic molecule, i.e contains both hydrophobic and hydrophilic parts. However, these results might not be representative due to bad statistics. Secondly, an experiment ran at BESSY II at Helmholtz-Zentrum Berlin using X-ray Photoelectron Spectroscopy in liquids. In this experiment, Lauryldimethylamine oxide (LDAO) was used instead of Trimethylamine N-oxide (TMAO) due to some practical reasons. The behaviour of urea and LDAO molecule was studied when these molecules were in different and same solutions. The purpose of this experiment is to find out the mechanism of LDAO against urea. Finally, LDAO interacts with urea and a possible mechanism between them is suggested.
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Ventura, Marcello. "Prion protein: does N-terminal domain allow vesicular micronutrients uptake?" Doctoral thesis, Università degli studi di Padova, 2009. http://hdl.handle.net/11577/3426867.
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Prion protein (PrPC) is a cell surface glycoprotein, anchored by the GPI to the cell membrane. It seems to be involved in some neurodegenerative diseases, but its physiological function is still undefined. Many cellular functions were proposed as PrPC roles, such as the modulation of several signal transduction pathways known to promote cellular survival and the protection against oxidative stress. Nevertheless, the main hypothesis on PrPC role is in copper homeostasis. Copper is an essential micronutrient (EMN) and all EMNs (vitamins and metals) are present in bodies and foods in micromolar concentrations. This feature can easily lead to deficiency. It is also clear that deficiency of EMNs produces overlapping diseases symptoms. Also the uptake pathway (endocytosis) is common for many EMN. Thanks to these reasons we hypothesize that prion protein could allow EMNs uptake. The aim of this work is to study these binding processes and their characteristics. So N-terminal domain of mouse PrPC (mPrP23-109) was recombinant expressed and purified. To study specificity and characteristics of metals binding process, mPrP23-109 was titrated with five first transition serie divalent metals (Mn, Co, Ni, Cu, Zn), at several pH values and it was followed in fluorescence spectroscopy. Furthermore, to define if metal binding could drive structural rearrangements in the prion protein N- terminal domain, we performed structural analysis using circular dicroism (CD). To investigate vitamins stacking between triptophan indolic rings, mPrP23-109 was titrated with four vitamins belonging to B group and titrations were monitored by fluorescence anisotropy, that allows to know the fluorophores average molecular rotational speed. Finally, we also investigated the possibility that prion protein N- terminal domain could interact with membrane mimetic systems. Our findings confirm that prion protein has a functional role in copper homeostasis. We also propose that prion protein, together with copper, plays a key role in integrated endocytic uptake pathway, involving all EMNs.La protein prionica (PrPC) è una glicoproteina legata alla superficie extracellulare della membrana tramite l’ancora GPI. Ad essa è stata attribuita la causa di alcune malattie neuro degenerative, ma la sua funzione fisiologica non è ancora stata definita. Molte funzioni sono state proposte, tra cui la modulazione di alcune vie di transduzione del segnale che promuovono la sopravvivenza cellulare e la protezione da stress ossidativo, ma l’ipotesi principale è una funzione nell’omeostasi del rame. Il rame è un micro nutriente essenziale (MNE) e tutti gli MNE sono presenti nel corpo e negli alimenti in concentrazioni micromolari; ciò può determinare facilmente una deficinza. È ormai chiaro che i sintomi da deficienza sono comuni a molti MNE. Inoltre anche le vie di assorbimento di molti MNE sono analoghe fra loro, usando vie endocitiche. Grazie a queste evidenze abbiamo ipotizzato che la PrPC possa essere coinvolta nell’assorbimento degli MNE. Lo scopo di questo lavoro è di studiare questi legami e le loro caretteristiche. Per far ciò, è stato espresso e purificato il dominio N-terminale della proteina prionica di topo (mPrP23-109). Per studiare la specificità e le caratteristiche del legame con i metalli, la proteina ricombinante è stata titolata con cinque metalli divalenti della prima serie di transizione (Mn, Co, Ni, Cu, Zn), a diversi valori di pH utilizzando la spettroscopia di fluorescenza. Per definire se le interazioni osservate fossero funzionali, è stata effettuata l’analisi strutturale in dicroismo circolare. Per verificare la proabile interazione tra le vitamine ed i triptofani della proteina, mPrP23-109 è stata titolata con quattro vitamine, appartenenti al gruppo B, ed è stata monitorata la velocità dei fluorofori mediante l’anisotropia di fluorescenza. Infine è stata anche studiata l’interazione tra dominio N-terminale della PrPC con sistemi mietici di membrane. I nostri risultati confermano che la PrPC ha un ruolo funzionale nell’omeostasi del rame. Inoltre proponiamo che la proteina prionica, insieme con il rame, ricopre un ruolo chiave nell’assorbimento integrato degli MNE, via endocitosi.
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Sanchez, Perez Maria Concepcion. "Study of the N-terminal domains of MDM2 and MDM4, and their potential for targeting by small-molecule drugs." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/8763.
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The MDM2 and MDM4 oncoproteins are both involved in regulating the tumour suppressor, p53. While the MDM2–p53 interface is structurally and biophysically well characterised, the MDM4-p53 interaction has only recently attracted researchers’ attentions. The goal of this project was to establish structural and chemical ground rules for the disruption of the interactions between the N-terminal domains of MDM2/4 and p53, which is an attractive anticancer strategy. In the current work, successful recombinant production and purification protocols for both the N-terminal domains of MDM2 (i.e. MDM2-N, residues 11-118) and MDM4 (MDM4-N, residues 14-111) have been established, yielding protein in sufficient quantity and quality for analysis using nuclear magnetic resonance spectroscopy (NMR). Two screening strategies were employed to identify small-molecule antagonists of the MDM2-N:p53 interaction. First, a virtual screening exercise identified several compounds that were shown (by NMR) to bind to MDM2-N with μM KDs. Docking studies supported by NMR chemical shift perturbation analysis suggested proposals for binding modes. The results are discussed in relation to the previously reported binding to MDM2-N of well-characterised inhibitors of the MDM2:p53 interaction such as Nutlin-3. Second, a fragment-based library was screened against MDM2-N using TROSY-type NMR spectra to monitor binding. Several hits were identified and the results are discussed with regard to the “druggability” of the MDM2-N p53 interaction. To better understand the p53-binding groove of MDM4-N, multidimensional NMR was used to investigate the structure and backbone dynamics of double-isotopically labelled samples of MDM4-N, both free (i.e. apo-MDM4-N) and in complexes with a p53-derived peptide or Nutlin-3. The apo-MDM4-N is more conformationally dynamic than MDM2, since it contains unstructured regions. These regions appear to become structured upon binding of a ligand. MDM4 appears to bind its ligand through conformational selection and/or an induced fit mechanism involving reorganization of key sub-sites within the binding groove. This study highlighted Abstract differences between Nutlin-3 and peptide binding that suggest the rational design of specific inhibitors of the MDM4:p53 interaction.
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McNeil, Gerard P. "Characterization of DNA-Protein Interactions at the NT/N Promoter: Proles for AP-1 and ATF Proteins." eScholarship@UMMS, 1996. http://escholarship.umassmed.edu/gsbs_diss/269.
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The focus of experiments presented in this dissertation is to determine how signals created by exposure to environmental stimuli are integrated at the level of transcription, resulting in the generation of specific patterns of gene expression. The model system used was expression of the neurotensinl neuromedin N (NT/N) neuropeptide gene in the neuroendocrine PC12 cell line. This gene is synergistically activated in PC12 cells in response to nerve growth factor, lithium, glucocorticoids, and activators of adenylate cyclase. Several cis-regulatory elements were identified within a 200 bp regulatory region, including AP-1, CRE, and GRE-like elements. Mutational analysis confirmed the importance of these elements for responses to inducer combinations. The primary objective was to identify proteins that interact with NT/N promoter sequences and determine if they are important in mediating responses to inducer combinations.
The first set of experiments was designed to investigate changes in AP-1 binding activity. Previous analysis had shown that mutation of the AP-1 site severely curtails responses to all inducer combinations indicating that AP-1 plays a pivotal role in NT/N gene activation. DNA binding studies using in vitro synthesized AP-1 proteins revealed that all heterodimeric combinations could bind both the AP-1 and JARE sites; however, these complexes displayed a higher affinity for the AP-1 site. c-Jun homodimers were also found to bind both these sites albeit with a lower affinity and with a preference for the JARE site. These studies revealed that specificity is probably not at the level of DNA binding. Therefore, it was possible that only a subset of AP-1 proteins were activated upon stimulation. DNase I footprint analysis using nuclear extracts from PC12 cells showed changes in protection at the consensus AP-1 site upon treatment with inducers suggesting changes in AP-1 binding activity. It was found that AP-1 binding activity was increased upon stimulation, with the major component being Jun B. However, substantial levels of c-Fos and c-Jun were also detected at some time points. These results coupled with transfection data demonstrating that forced expression of c-Jun and c-Fos result in potent synergistic activation of the NT/N promoter support the hypothesis that c-Jun and c-Fos are also involved in NT/N gene activation.
DNase I footprinting studies using PC12 nuclear extracts also revealed substantial areas of protection surrounding the CRE element. This result, along with the high degree of conservation of these sequences between human and rat, suggested they play a role in the regulation of the NT/N gene in PC12 cells. Mutational analysis of this region showed that sequences upstream of the CRE were important for full activation of the NT/N promoter. Specific mutation of the CRE resulted in a 75% decrease in activity upon induction, a level similar to that observed previously with less precise linker scanner mutations. This site had also been shown to be critical for c-Jun mediated NT/N activation, even though c-Jun homodimers do not bind this site in vitro. Therefore, nuclear extracts from PC12 cells were tested for the presence of proteins which could bind this site. Complexes composed of both c-Jun and ATF-2 were found in extracts from both uninduced and induced PC12 cells. ATF-2 could mediate both the recruitment of c-Jun to this site as well as mediate the effect of activators of adenylate cyclase, since ATF-2 has been shown to be a target for protein kinase A in vitro. Expression of ATF-2 in PC12 cells resulted in a modest increase in NT/N promoter activation. The significant levels of endogenous ATF-2 protein in PC12 cells most likely accounts for the relatively small magnitude of this effect. Experiments with the closely related protein, ATF-a2, revealed that it potently antagonizes c-Jun activation while forced expression of ATF-2 did not affect c-Jun activation under the conditions analyzed. Therefore, ATF proteins could be involved in both activation and repression of the NT/N gene. Both c-Jun and ATF-2 have been shown to be activated by c-Jun N-terminal kinase (JNK) in response to environmental stress or cytokine activation. Therefore, the ability of inducers to activate the previously described N-terminal ATF-2 activation domain was investigated using a GAL4-ATF-2 (1-109) chimer construct. This construct was not significantly activated by inducer combinations that result in high level NT/N gene expression, indicating that activation of ATF-2 through this pathway is not involved in NT/N gene activation. Also activation of JNK, a MAPK which activates both c-Jun and ATF-2, only partially substituted for NGF indicating that NGF activates an additional pathway. The data presented here support a model involving synergistic transcriptional activation of the NT/N promoter by c-Jun/c-Fos, ATF-2, ATF-2/c-Jun and the GR. ATF-2 was found to enhance NT/N promoter activation while a splice variant (ATF-2 195) lacking a central portion of ATF-2 that is rich in Ser/Thr residues had no effect suggesting that this region could be important for ATF-2 activation in PC12 cells. The identification of the signaling pathways that mediate the effects of inducer combinations on NT/N gene activation will be an important future goal and should provide insights into the control of neuronal gene expression.
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Popham, Jennifer Mei-An. "A solid state NMR dipolar recoupling study of surface interactions of a N-terminal statherin fragment bound to hydroxyapatite /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/8517.
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Salek, Reza M. "NMR studies of the heat shock protein 90 N-terminal domain." Thesis, University College London (University of London), 2005. http://discovery.ucl.ac.uk/1446807/.
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The Hsp90 based chaperone is a ubiquitous protein-folding system in the cytoplasm of eukaryotes. Several signal transduction systems and cell cycling pathways utilise an interaction with Hsp90 as an essential component. The Hsp90 chaperone is an ATP dependent chaperone, which is active as a dimer. The N- terminal domain of Hsp90 itself has very weak ATPase activity and plays an essential role in the mechanism of dimerisation. This study attempts to elucidate the nucleotide binding effects on the Hsp90 N-terminal domain by NMR. Accomplishing backbone assignments of the apo- and AMP-PNP bound forms provided a system for which individual residues could be investigated. About 200 backbone amide peaks in the HSQC were observed out of a reported 207 residues for the Hsp90 N-terminal domain. Out of these, 192 for the apo- and 182 for the AMP-PNP bound forms were assigned. Assignments were also obtained for the HN, N, C, and CO nuclei. Comparison of apo and AMP-PNP HSQC spectra showed large shift differences in areas where the nucleotide binds and in a conserved loop, which has been proposed to act as a lid to the active site. The chemical shift pattern of the AMP-PNP bound form compared to that of the ADP bound form showed a different local environment for at least 30 residues, suggesting that different nucleotide bound conformations are more than just nucleotide structural differences. Analysing the NMR results suggests that binding of AMP-PNP to the Hsp90 N-terminal domain is not sufficient to cause lid closure as previously thought. Relaxation studies highlighted regions that had different local motions in the apo- and nucleotide bound form based on individual residues within the Hsp90 N- terminal domain. The protein rotational correlation time was measured at 12.5 ns in the apo and 14 ns in the AMP-PNP bound form. No interaction between the labelled N-terminal domain and non-labelled middle domain of Hsp90 was observed. The antibiotic, novobiocin, which inhibits other members of the GHKL superfamily, was also shown not to bind to the N-terminal domain, consistent with previous studies. The study of two mutants, A107N and T101I, showed the effects of mutation in the lid region of the N-terminal domain causing chemical shifts of between 20-30 residues. Neither of these two mutants were able to bind AMP-PNP. In all nucleotide bound X-ray crystal structures solved to date, no difference between the ADP and ATP bound forms has been observed and only one conformation was found. The results presented here suggest that the structure in solution is much more variable than previously envisaged.
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Patel, Avnish. "Studies of the intrinsically disordered N-terminus of murine prion protein." Thesis, University of Reading, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.577771.
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Mammalian prion protein is able to cause a multitude of neurological maladies, most notably the transmissible spongiform echephalopathies. All are characterised by the misfolding of a non-pathogenic form the cellular protein Prpc to a misfolded aggregated isoform Prpscwhich leads to neurotoxicity and apoptosis characterised by brain spongiosis and amyloid plaque deposits. The exact molecular mechanisms of pathogenicity remain unknown for both the transmissible and inherited forms of disease. An artificially generated deletion of residues 105-125 of Prpc, encoding largely hydrophobic amino acids, in the intrinsically disordered N-terminus of the protein is hig~ly neurotoxic and has been shown to mediate a TSE like phenotype when expressed in transgenic mice. A possible mechanism of action consistent with prion protein exhibiting varying isoforms is that the deletion mutant may fold to a structure comparative to that of a naturally occurring pathogenic form. Biochemical and biophysical characterisation of the deleted form of Prpc could support this possibility. To explore the role of residues 95-135 in protein folding an extended set of deletion mutants of the hydrophobic region were created and the encoded Prpc like proteins expressed and purified. Among the mutants made it was found that deletion of solely residues 105-125 within the hydrophobic region gave rise to subtle structural alterations when compared to parental Prpc. As Prpc is a GPI anchored protein normally associated with a membrane environment, membrane interaction assays were also performed with deletion mutants revealing that that a charge cluster and hydrophobic region acted synergistically to bind, insert into and disrupt anionic membranes. These findings did not support a mechanism of action for the deletion ~105-125 mediating toxicity through membrane pore formation but rather a subtle structural change that may be consistent with a receptor 4 mediated toxicity model. Possible further cellular and structural studies with defined deletions of Prpc with a view to deciphering the neurotoxic mode of action are discussed. 5
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Wong, Shian Yea. "Identifying protein interaction partners of the Pitx2c N-terminus during embryogenesis." Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=123305.
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The vertebrate body is patterned asymmetrically along the left-right axis. Left-right patterning is required to establish correct asymmetric organ formation and positioning, and ultimately it is essential for normal physiological functioning. Misregulation in this process can lead to severe physiological defects in multiple organs, including the heart and gut. Pitx2c is a paired-like homeodomain transcription factor and is crucial for translating left-right signals into asymmetric morphogenesis. It is asymmetrically expressed in the left lateral plate mesoderm and continues to be expressed on the left side of organ primordia that are destined to become asymmetrically formed or positioned organs. Previous work studying Pitx2c has demonstrated its evolutionarily conserved role in left-right patterning. While the homeodomain is important for Pitx2c's function, our lab identified a putative interaction domain in the N-terminus of Pitx2c that is required for its function in left-right patterning. To further characterize the role of Pitx2c, I used a yeast two-hybrid screen to identify candidate protein interaction partners of its N-terminus. Thirty-two candidates were identified from the screen. Candidates that were isolated at least twice were prioritized for further analysis. After eliminating non-coding clones, false positive interactions, and confirming interactions of the candidate putative interaction domains with the Pitx2c N-terminus in yeast, six candidates remained: Anti-silencing function 1 homolog B (S. cerevisiae; Asf1b), Eukaryotic translation initiation factor 3, subunit A (Eif3a), Eukaryotic translation initiation factor 3, subunit M (Eif3m), Niemann-Pick C type 2 (Npc2), Serine/arginine-rich splicing factor 2 (Srsf2), and Serine/arginine-rich splicing factor 15 (Srsf15). mRNA Expression patterns of these six Pitx2c candidate protein interaction partners was analyzed by whole mount in situ hybridization and compared to Pitx2c expression patterns. All candidates were expressed symmetrically in the embryo, including tissues where Pitx2c is asymmetrically expressed. In the chick embryo, Npc2 and Srsf2 were expressed in a tissue-specific manner from HH stage 10 to HH stage 26, while Eif3a and Eif3m were broadly expressed throughout the embryo from HH stage 10 and only became enriched in particular tissues at HH stage 24 to HH stage 26. All candidates were coexpressed with Pitx2c in the looped heart tube. Only Srsf2 was coexpressed with Pitx2c in the lateral plate mesoderm. The known functions of these candidates are consistent with the known role of Pitx2c in transcriptional regulation and suggests new roles for Pitx2c in mRNA processing and translational regulation during left-right patterning.Le corps des vertébrés est structuré asymétriquement le long de l'axe gauche-droit. La latéralité est requise afin d'établir la formation des organes ainsi que leur positionnement qui, ultimement, sont essentielles pour un fonctionnement physiologique normal des vertébrés. Lors de ce processus, une mauvaise régulation peut causer des troubles physiologiques sévères dans plusieurs organes incluant le cœur et l'intestin.Le facteur de transcription homéodomaine Pitx2c joue un rôle essentiel lors de la traduction des signaux gauche-droit en morphogénèse asymétrique des organes. Pitx2c est exprimé de façon asymétrique dans le mésoderme de la plaque latérale gauche et continu d'être exprimé du côté gauche des futurs organes asymétriques. Quelques études ont démontré que Pitx2c a un rôle qui est conservé lors de l'évolution dans la latéralité. Le rôle de l'homéodomaine est important pour la fonction de Pitx2c et nous avons démontré dans notre laboratoire qu'un domaine d'interaction de la partie N-terminale de Pitx2c est aussi important pour sa fonction lors de la latéralité.Afin de caractériser d'avantage le rôle de Pitx2c, j'ai utilisé la méthode de double hybride afin d'identifier des protéines candidates d'interaction avec la partie N-terminale de Pitx2c. Trente-deux candidats ont été sélectionnés. De ce nombre, les faux positifs ainsi que les candidats qui représentent région la non-codante d'une protéine ont été supprimés ce qui a permis de réduire la liste à six candidats : Anti-silencing function 1 homolog B (S. cerevisiae; Asf1b), Eukaryotic translation initiation factor 3, subunit A (Eif3a), Eukaryotic translation initiation factor 3, subunit M (Eif3m), Niemann-Pick C type 2 (Npc2), Serine/arginine-rich splicing factor 2 (Srsf2), ainsi que Serine/arginine-rich splicing factor 15 (Srsf15).Les patrons d'expression des six protéines candidates potentielles d'interaction avec Pitx2c ont été examinés par hybridation in situ et ont été comparé avec le patron d'expression de Pitx2c. Chez tous les candidats, l'expression asymétrique dans les embryons de poulet a été observée incluant dans les tissus où Pitx2c est aussi exprimé asymétriquement. Npc2 et Srsf2 sont exprimés de façon spécifique dans certains tissus à partir du stade HH10 chez le poulet. Eif3a et Eif3m sont largement exprimés dans les embryons à partir du stade HH10 mais leur expression est enrichie dans certains tissus à partir du stade HH22. L'expression de tous les candidats coïncide avec celle de Pitx2c dans la courbure du tube cardiaque. Seulement l'expression de Srsf2 coïncide avec celle de Pitx2c dans le mésoderme de la plaque latérale gauche. Les fonctions des différents candidats suggèrent que ces partenaires potentiels d'interactions de la partie N-terminale de Pitx2c pourraient jouer un rôle important lors de la régulation de la transcription, la formation de l'ARN messager et la régulation de la traduction lors de la latéralité.
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Edwards, M. C. "Studies on (ADP-ribose)sub(n)-protein conjugates synthesised in vitro." Thesis, University of Reading, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.371438.
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Mao, Yunfei. "Substrate Recognition and Mechanistic Studies of Protein N-Terminal Methyltransferase 1." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4414.
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The methylation at the α-N-terminal amines of proteins that start with a canonical motif X-P-K (X=A/P/S) has been a known modification for nearly four decades. In 2010, protein α-N-terminal methyltransferase 1 (NTMT1/NRMT1) was identified as the first enzyme responsible for this modification. NTMT2 was discovered as a second member belonging to this family, but it was reported as a mono-methylase. The identification of RCC1, retinoblastoma (Rb) protein, centromere protein-A/B (CENP-A/B), and DNA damaged-binding protein 2 (DDB2) as new NTMT1 substrates revealed NTMT1’s biological significance in mitosis, cell-cycle regulation, centromere formation, and damaged DNA repair, respectively. Although significant progress had been made, a clear understanding of how NTMT1 recognizes substrates remains to be determined. Also, there is no specific small molecule inhibitor for NTMT1.
To fill these gaps, we first established a fluorescence-based assay for kinetic characterization of NTMT1. Subsequently, ternary complex crystal structures of NTMT1 were obtained to illustrate the structural basis for enzyme-substrate interactions. The structures of the enzyme-substrate complex coupled with mutagenesis, binding, and enzymatic studies demonstrated the key elements involved in interaction with its substrates. In the meantime, we utilized computational studies and fluorescence assays for novel small molecule discovery. Lastly, we closely monitored the substrates’ methylation progression by NTMT1 and NTMT2 in parallel using a MALDI-MS based assay.
Our results indicated that NTMT1 follows a Bi-Bi mechanism, and its methylation proceeds in a distributive pattern. Furthermore, NTMT1 was identified has broad substrate specificity beyond its canonical motif X-P-K (X=A/P/S), since X can be any amino acid except D/E and the third amino acids can also be R. We had also discovered an inhibitor that targets the substrate binding site of NTMT1 with IC50 = 7 µM. Lastly, our methylation progression studies has demonstrated that NTMT2 can also di-, tri-methylate certain substrates although its methylation rate is lower than NTMT1.
Overall, this project has laid the foundation for further investigation of N-terminal methylation in terms of functions, mechanisms, and inhibitor design.
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Copin, Nane. "Catecholamine synthesising enzymes in the programming of hypertension by mild protein restriction during gestation." Thesis, University of Southampton, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268370.
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Orton, Christopher R. "Analysis of Protein Adduction Kinetics and the Effects of Protein Adduction on C-Jun N-Terminal Kinase Signaling." Diss., The University of Arizona, 2006. http://hdl.handle.net/10150/194247.
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Defining the mechanics and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. In this dissertation I describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein using quantitative mass spectrometry. Adducts are formed by electrophiles at Cys-14 and Cys-47 on the metabolic enzyme glutathione-S-transferase P1-1 and accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples. This method was used to measure rate constants for adduction at both positions with two different model electrophiles, IAB and BMCC. The results indicate that Cys-47 was approximately 2-3-fold more reactive toward both electrophiles than was Cys-14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system. Quantitative analyses of protein modifications provide a means of determining the reactivity and selectivity of damaging protein modifications in chemical toxicity.Another area of study explored in this dissertation is looking at the effects of protein alkylation on activating cellular signaling pathways, specifically the JNK signaling pathway. Protein adduction has been shown to be selective between different alkylating agents. It would then be reasonable to think this selectivity of adduction translates to selectivity of downstream consequences or cellular events directly tied to specific adductions. My work will show how treatment of HEK293 cells with either IAB or BMCC leads to differences in activation of JNK signaling. In addition, I've been able to show a difference in selectivity of a number of adducted targets by each alkylating agent, which are directly involved in regulation of the JNK signaling pathway. These studies illustrate not only the significance of protein adduction, but the importance for continual research to better understand their behavior in living systems.
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Watson, Joanna. "Structural and biochemical insight into the interactions of Cdc42 with TOCA1 and N-WASP." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/268520.
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Cdc42 is a member of the Rho family of small GTPases, which, together with its homologues RhoA and Rac1, controls a multitude of cellular functions via the actin cytoskeleton. Cdc42 exerts its effects on the cytoskeleton via effector proteins of the Wiskott-Aldrich Syndrome (WASP) family and the Transducer of Cdc42-dependent Actin assembly (TOCA) family. The WASP family and their activation by Cdc42 have been thoroughly studied in vitro and are well understood. Conversely, understanding of the TOCA family remains limited by a lack of biochemical, biophysical and structural insight. An investigation of the TOCA1-Cdc42 interaction is described here, revealing a relatively low affinity interaction with a dissociation constant in the micromolar range. This is 10-100x weaker than other Rho-effector interactions and suggests that TOCA1 must first be co-localised with Cdc42 to achieve stable binding in vivo. The solution NMR structure of the Cdc42 binding HR1 domain of TOCA1 provides the first structural data on this protein and reveals some interesting structural features that may relate to binding affinity and specificity. A structural model of the Cdc42-HR1 complex provides further insight into differential specificities and affinities of GTPase-effector interactions. NMR and actin polymerisation assays provide insight into the pathway of Cdc42/TOCA1/WASP-dependent actin assembly, suggesting unidirectional displacement of TOCA1 by N-WASP. A comparison of the Cdc42- TOCA1 model with an NMR structure of Cdc42 in complex with the GTPase binding domain of WASP reveals a possible mechanism by which an ‘effector handover’ from TOCA1 to N-WASP could take place. Small GTPases such as Cdc42 are lipid modified and membrane anchored via their C- termini in vivo, so in vitro studies using truncated, unmodified GTPases are limited in their biological interpretation. This project also aimed to develop methods to study full length and membrane-anchored GTPases in vitro. Lipid modified protein was produced, which showed a weak affinity for liposomes, and so structural studies of membrane anchored protein are within reach. Further method development is now required to achieve stable membrane anchoring of lipid modified GTPases for detailed NMR studies.
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Cain, Joel Aaron. "Evaluating the Relationship Between N-Glycosylation and Protein Stability in Campylobacter jejuni." Thesis, The University of Sydney, 2021. https://hdl.handle.net/2123/25679.
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The major enteric pathogen Campylobacter jejuni was the first prokaryote demonstrated to perform N-linked protein glycosylation. Although protein post-translational modifications involving glycan attachment to proteins are strongly associated with virulence, the mechanisms and functions of N-glycosylation remains to be determined.
This thesis aimed to address some of the outstanding questions regarding the role of N-glycosylation in C. jejuni. Through application of quantitative proteomics we assessed the global effect of N-glycosylation to establish a causal relationship between the proteome and pgl- associated phenotypes. We also applied a multi-protease approach to build upon previous identification of the C. jejuni N-glycoproteome. In doing so, we developed a strategy that exploited the proteome of pgl- negative bacteria to identify novel occupied and unoccupied sequons and to qualitatively assess site stoichiometry.
In expanding our analysis to other pgl-negative strains we established that loss of the modification is strongly associated with a transcription- independent reduction in glycoprotein abundance. Using N- degradomics approaches based on N-terminal amine isotopic labelling of substrates (N-TAILS), we demonstrated this effect is consistent with increased proteolytic activity towards N-glycoproteins. Furthermore, this activity is not apparent in proteins modified by an N-glycan. With the goal of identifying the responsible protease, we further characterised the putative C-terminal glycoprotein protease, Cj0511. Through observations of increased proteolysis and compensatory elevation of alternative proteases in response to the loss of cj0511 we demonstrated that Cj0511 is likely an important mediator of protein homeostasis in vivo.
This thesis has confirmed that N-glycosylation is an important contributor to C. jejuni biology by protecting disordered and exposed structural elements of proteins from self-proteases in the protease-rich C. jejuni periplasm.
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Schulz, Benjamin Luke. "Analysis and mechanisms of site-specific N-linked protein glycosylation by oligosaccharyltransferase /." Zürich : ETH, 2008. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17559.
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Kotecha, Suhas Ashok. "G-protein coupled receptor modulation of N-methyl-D-aspartate channel activity." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/NQ63766.pdf.
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Yadao, Franeli M. (Franeli Marie). "Physical interaction between human b-N-acetylhexosaminidase A and its activator protein." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=24049.
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GM$ sb2$ ganglioside hydrolysis requires the formation of a ternary complex consisting of substrate, enzyme ($ beta$-N-acetylhexosaminidase A = Hex A), and the GM$ sb2$ activator protein. In order to study the interaction between Hex A and GM$ sb2$ activator, the human GM$ sb2$ activator cDNA was cloned into the p-FLAG vector. The fusion protein (FLAG-AP) was expressed in E. coli, and purified to homogeneity using immunoaffinity chromatography.A retardation assay was designed using the immunoaffinity column to detect transient interactions between FLAG-AP and Hex A. Hex A and Hex S are retarded by the column, but not Hex B or unrelated proteins. Hex A retardation is absolutely dependent upon the presence of immobilized FLAG-AP, but does not require the presence of GM$ sb2$ ganglioside. Interaction of GM$ sb2$ activator and Hex A does not involve the enzyme's active site, but does appear to depend upon hydrophobic interactions between the two proteins.
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Wang, Jian, and 王健. "Identification and characterization of N-terminal kinase like protein in hepatocellular carcinoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47036370.
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Bratzel, Graham Hayden. "Sequence-structure correlations in the MaSp1 protein of N. clavipes dragline silk." Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/67610.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2011.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 77-86).
Silk is a hierarchically structured protein fiber with exceptional tensile strength and extensibility, making it one of the toughest and most versatile biocompatible materials. While experimental studies have shown that the molecular structure of silk has a direct influence on the stiffness, toughness, and failure strength of silk, few molecular-level analyses of the nanostructure of silk assemblies, in particular under variations of genetic sequences, have been published. Here, atomistic-level structures of wildtype as well as modified MaSp1 protein from the N. clavipes spider dragline silk sequences are reported, obtained using an in silico approach based on replica exchange molecular dynamics (REMD) and explicit water molecular dynamics. In particular, the atomistic simulations discussed in this parametric study explore the effects of the poly-alanine length of the N. clavipes MaSpi peptide sequence, solvent conditions, and nanomechanical loading conditions on secondary and tertiary structure predictions as well as the nanomechanical behavior of a unit cell of 15 strands with 900-1000 total residues used to represent a cross-linking 7-sheet crystal node in the network within a fibril of the dragline silk thread. Understanding the behavior of this node at the molecular scale is critical for potentially bypassing strength limits at this length scale and vastly improving silk for medical and textile purposes as well as synthetic elastomers and polymer or aramid fiber composites with a similar molecular structure and noncovalent bonding for aerospace, armor, and medical applications. The main hypothesis tested is that there exists a critical minimum length of the poly-alanine repeat that ensures the formation of a robust cross-linking the [beta]-sheet crystal. Confirming earlier experimental and computational work, a structural analysis reveals that poly-alanine regions in silk predominantly form distinct and orderly [beta]-sheet crystal domains while disorderly regions are formed by glycine-rich repeats that consist of 310-helix type structures and 7-turns. These predictions are directly validated against experimental data based on dihedral angle pair calculations presented in Ramachandran plots combined with an analysis of the secondary structure content. The key results of this study are: e A strong dependence of the resulting silk nanostructure on the poly-alanine length. The wildtype poly-alanine repeat length of six residues defines a critical minimum length that consistently results in clearly defined [beta]-sheet nanocrystals allowing for misalignment. For poly-alanine lengths below six residues, the /-sheet nanocrystals are not well-defined or not visible at all, while for poly-alanine lengths above six the characteristic nanocomposite structure of silk emerges with no significant improvement of the quality of the sheet nanocrystal geometry. A simple biophysical model is presented that explains the minimum length scale based on the mechanistic insight gained from the molecular simulations. The efficient stacking of the [beta]-sheets of a well-defined crystal reinforces local hydrophobicity and prevents water diffusion into a crystal above a critical size. Nanomechanical testing reveals that the combination of the 12-alanine length case and central pull-out loading conditions results in delayed failure by employing a hierarchy of strong [beta]-sheets and soft, extensible semi-amorpous regions to overcome a predicted H-bond saturation. This work constitutes the most comprehensive study to-date of the molecular structure prediction and nanomechanical behavior of dragline silk. Building upon previous computational studies that used similar methods for structure prediction and mechanical analysis, e.g. REMD and force-control loading, this work presents: the first results of the near-native structures determined by REMD after equilibration in TIP3P explicit solvent, the first parametric study of the effects of modifying the wildtype poly-alanine segment length to values outside the range naturally observed for MaSp1 on structure prediction and nanomechanical behavior, and, the first comparison between previously published loading conditions, i.e. the Stretch test, and the novel Pull-out loading conditions that are hypothesized to be more appropriate for modeling of the in situ loading of the cross-linking [beta]-sheet crystal. Further parametric studies in peptide sequence to optimize bulk fiber properties must involve changes in simulated nanomechanical loading conditions to properly assess the effects of the changes in peptide sequence. These findings set the stage for understanding how variations in the spidroin sequence can be used to engineer the structure and thereby functional properties of this biological superfiber, and present a design strategy for the genetic optimization of spidroins for enhanced mechanical properties. The approach used here may also find application in the design of other self-assembled molecular structures and fibers and in particular biologically inspired or completely synthetic systems.
by Graham Hayden Bratzel.
S.M.