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1
Chatani, Eri. "The Structural Basis for the Functionality and Stability of Bovine Pancreatic Ribonuclease A". Kyoto University, 2002. http://hdl.handle.net/2433/149886.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第9594号
農博第1222号
新制||農||840(附属図書館)
学位論文||H14||N3626(農学部図書室)
UT51-2002-G352
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 林 力丸, 教授 關谷 次郎, 教授 西岡 孝明
学位規則第4条第1項該当
2
Fujii, Takahiro. "Studies of structural formation of bovine pancreatic ribonuclease A : Role of the carboxyl terminal region". Kyoto University, 2000. http://hdl.handle.net/2433/151623.
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本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものであるKyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第8628号
農博第1155号
新制||農||814(附属図書館)
学位論文||H12||N3473(農学部図書室)
UT51-2000-R34
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 林 力丸, 教授 清水 昌, 教授 岩村 俶
学位規則第4条第1項該当
3
Tanimizu, Naoki. "Conformational properties of amino acid residues in the active site of bovine pancreatic ribonuclease A". Kyoto University, 1999. http://hdl.handle.net/2433/181366.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第7960号
農博第1069号
新制||農||785(附属図書館)
学位論文||H11||N3294(農学部図書室)
UT51-99-M265
京都大学大学院農学研究科農芸化学専攻
(主査)教授 林 力丸, 教授 池田 篤治, 教授 岩村 俶
学位規則第4条第1項該当
4
Council, Claire E. "Evaluation of sequential chemoselective peptide ligation and molecular dynamics simulations as tools for the total synthesis of proteins: an example using bovine pancreatic ribonuclease A". Thesis, University of Surrey, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.583341.
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Chemoselective ligation between two peptides can be used to produce synthetic peptides and proteins that cannot easily be synthesised as single peptides by solid phase peptide synthesis. Chemoselective reaction between an aldehyde and hydrazine or hydroxylamine, masking the aldehyde as a 1, 2-amino alcohol, has been investigated as a method of sequential ligation that will allow synthesis of individual peptides in high yield and purity, enabling more than two peptides to be ligated. Protected amino acids were used as precursors for the synthesis of 1, 2-amino alcohol derivatives that could be used in solid phase peptide synthesis for the production of peptides bearing a C-terminal 1, 2-amino alcohol, as a masked aldehyde for use in ligation reactions. Limitations of this method led to alternative investigations, using peptides bearing an N-terminal serine as a masked aldehyde, and a C-terminal hydrazide. Bovine pancreatic ribonuclease A was chosen as an example protein for sequential ligation and using this method, peptides were synthesised in high yield and purity for use in ligation reactions. Trial ligation reactions with short test peptides were performed successfully, however problems were experienced during ligations using peptide fragments of ribunuclease due to involvement of the cysteine side chains. Methods to overcome these unwanted reactions resulted in insoluble peptide fragments. Computer modelling using molecular dynamics simulations has been used to investigate the effect of replacing native peptide bonds in ribonuclease on the structure of the protein. The method of molecular dynamics simulation was validated through comparison of the structure of a mutant of ribonuclease from experimental NMR data to the structure produced after a molecular dynamics simulation. Results of the modelling simulations suggest that replacement of native peptide bonds with the chemoselective bond formed through reaction of an aldehyde and hydrazine will have only minor implications for the structure of ribonuclease, and therefore should only have a small impact on enzyme activity .
5
Alade, Ayoade Nathaniel. "Investigating the Catalytic Role of Lysine Residue 41 in Pancreatic Ribonuclease A". Thesis, California State University, Long Beach, 2017. http://pqdtopen.proquest.com/#viewpdf?dispub=10603029.
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Understanding enzyme catalysis is one of the major goals in biology. Ribonuclease A (RNase A) is a key system to understanding protein structure and function provides an attractive system to investigate the catalytic role of active site interactions. Crystal structures show a lysine residue (Lys41) situated in the RNase A active site, and mutagenesis studies suggest this residue is important for catalysis. To evaluate the catalytic importance of the Lys41-phosphate interaction, double mutant cycle analysis was used. Individual mutation of lysine to arginine (K41R) and substitution of a phosphate oxygen with sulfur led to ∼350 and ∼100-fold decrease in kcat/KM, respectively. However, in the K41R background, substitution of the same oxygen with sulfur decreased activity by a similar amount (within 2-fold) as it did with the wild-type enzyme. This result provides evidence that functional interaction between Lys41 and the phosphate backbone of RNA substrates may not be solely limited between the two groups.
6
Bertrand, Jay Aaron. "X-ray crystal structures of inhibited bovine pancreatic trypsin". Diss., Georgia Institute of Technology, 1994. http://hdl.handle.net/1853/27321.
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Yuan, Chunhua. "Structural and conformational studies of bovine pancreatic phospholipase A2 /". The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487948807588005.
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8
Doherty, Aidan Joseph. "Studies on the sequence-selective nuclease, bovine pancreatic DNase I". Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359221.
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9
Tan, Kok Leong. "Protein engineering of bovine pancreatic deoxyribonuclease I by secreation of polypeptide elements". Thesis, University of Newcastle Upon Tyne, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285799.
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10
Dupureur, Cynthia M. "Structure-function studies in the active site of bovine pancreatic phospholipase A2 /". The Ohio State University, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487775034179833.
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11
Wang, Yingsong. "Investigating the In Vitro Oxidative Folding Pathways of Bovine Pancreatic Trypsin Inhibitor (BPTI)". FIU Digital Commons, 2013. http://digitalcommons.fiu.edu/etd/1029.
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The oxidative folding pathway of the disulfide containing protein bovine pancreatic trypsin inhibitor (BPTI) was one of the first to be elucidated and has served as a basis for understanding the folding pathways of other proteins. During the oxidative folding of reduced BPTI, two intermediates (N' and N*) accumulate in significant amounts and act as kinetic traps. Both N' and N* bury their two remaining free thiols in their hydrophobic cores, which inhibits further oxidation. Historically, the rate limiting step was considered to be the intramolecular rearrangements of N' and N* to another intermediate with two free thiols, NSH. The two free thiols in NSH are solvent-exposed and easily oxidized to a disulfide, producing native protein (N). Nevertheless, our research using reduced BPTI indicated that the folding rate of N* to N was proportional to the concentration of added glutathione disulfide (GSSG), inconsistent with the slow intramolecular rearrangement of N* to NSH. To confirm our initial results, the intermediate N* was purified and refolded in the presence of GSSG. The conversion of N* to N was dependent upon the disulfide concentration and singly mixed disulfide N*(SG) was observed during folding. These results emphasize that the folding of N* can proceed via a growth type pathway, direct oxidation of the two remaining thiols in N* by an exogenous small molecule disulfide, such as GSSG, to form N. Folding of reduced BPTI via N* was performed under changing concentrations of GSSG and GSH as a function of time. The folding was improved dramatically in terms of rate and yield.
Aromatic disulfides and thiols have been demonstrated to improve the folding efficiency of disulfide containing proteins including ribonuclease A (RNase A) and lysozyme. Herein, N* and N' were refolded in the presence of aromatic disulfides. Folding of the two kinetic traps with aromatic disulfides indicated that folding proceed via a growth type pathway. The singly and doubly mixed disulfide intermediates were observed during most folding reactions. The oxidative folding of reduced BPTI with aromatic disulfides and thiols were also investigated. Reduced BPTI can be folded to disulfide intermediates rapidly.
12
Staley, Jonathan Prescott. "Structural of early intermediates in the folding pathway of bovine pancreatic trypsin inhibitor". Thesis, Massachusetts Institute of Technology, 1993. http://hdl.handle.net/1721.1/43269.
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13
Liu, Xiaohong. "Structure-function relationship studies of bovine pancreatic phospholipase a2 and chicken muscle adenylate kinase /". The Ohio State University, 1996. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487936356159719.
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14
Kraunsoe, James A. E. "Inhibitors of serine proteinases". Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318814.
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15
Castro, Gallegos Jessica. "A human pancreatic ribonuclease variant kills cancer cells by apoptosis and reduces the expression of P-glycoprotein in MDR cell lines". Doctoral thesis, Universitat de Girona, 2010. http://hdl.handle.net/10803/7643.
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En aquesta tesi s'han estudiat les propietats antitumorals d'una variant de la ribonucleasa pancreàtica humana anomenada PE5 que incorpora un senyal de localització nuclear. Aquest estudi mostra que PE5 indueix l'apoptosi de les cèl·lules tractades i que aquesta mort és independent de l'activitat de p53. A més, l'efecte citotòxic no es veu afectat per un fenotip de resistència a múltiples drogues. Les dades també mostren que l'activitat citotòxica de PE5 és selectiva per a cèl·lules tumorals in vitro i que la capacitat citotòxica de les dues ribonucleases és semblant. S'ha estudiat l'efecte d'aquestes dues ribonucleases sobre el cicle cel·lular, l'activació de diferents caspases i l'expressió de proteïnes relacionades amb l'apoptosi i el cicle cel·lular. Els resultats indiquen que PE5 i l'onconasa maten les cèl·lules a través de mecanismes diferents. A més, PE5 però no l'onconasa, redueix l'acumulació de glicoproteïna-P en dues línies cel·lulars resistents a múltiples drogues.In this thesis the antitumor properties of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, have been investigated. This study shows that the cytotoxicity of PE5 is produced through apoptosis and that this ribonuclease does not require the activity of p53 to trigger the cell death. In addition, the cytotoxic effect is not prevented by a multiple drug resistance phenotype. The data also show that in vitro PE5 is selective for tumor cells and that PE5 and onconase induce cell death to the same extent. Effects of both ribonucleases on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins have been investigated. The results show that PE5 and onconase kill the cells through mechanisms with significant differences. In addition, PE5 but not onconase, reduces the accumulation of P-glycoprotein in two different multidrug-resistant cell lines.
16
Marahatta, Ram Prasad. "Folding of Bovine Pancreatic Trypsin Inhibitor (BPTI) is Faster using Aromatic Thiols and their Corresponding Disulfides". FIU Digital Commons, 2017. https://digitalcommons.fiu.edu/etd/3530.
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Improvement in the in vitro oxidative folding of disulfide-containing proteins, such as extracellular and pharmaceutically important proteins, is required. Traditional folding methods using small molecule aliphatic thiol and disulfide, such as glutathione (GSH) and glutathione disulfide (GSSG) are slow and low yielding. Small molecule aromatic thiols and disulfides show great potentiality because aromatic thiols have low pKa values, close to the thiol pKa of protein disulfide isomerase (PDI), higher nucleophilicity and good leaving group ability. Our studies showed that thiols with a positively charged group, quaternary ammonium salts (QAS), are better than thiols with negatively charged groups such as phosphonic acid and sulfonic acid for the folding of bovine pancreatic trypsin inhibitor (BPTI). An enhanced folding rate of BPTI was observed when the protein was folded with a redox buffer composed of a QAS thiol and its corresponding disulfide.
Quaternary ammonium salt (QAS) thiols and their corresponding disulfides with longer alkyl side chains were synthesized. These QAS thiols and their corresponding disulfides are promising small molecule thiols and disulfides to fold reduced BPTI efficiently because these thiols are more hydrophobic and can enter the core of the protein.
Conformational changes of disulfide-containing proteins during oxidative folding influence the folding pathway greatly. We performed the folding of BPTI using targeted molecular dynamics (TMD) simulation and investigated conformational changes along with the folding pathway. Applying a bias force to all atoms versus to only alpha carbons and the sulfur of cysteines showed different folding pathways. The formation of kinetic traps N' and N* was not observed during our simulation applying a bias force to all atoms of the starting structure. The final native conformation was obtained once the correct antiparallel β-sheets and subsequent Cys14-Cys38 distance were decreased to a bond distance level. When bias force was applied to only alpha carbons and the sulfur of cysteines, the distance between Cys14-Cys38 increased and decreased multiple times, a structure similar to the confirmation of N*, NSH were formed and native protein was ultimately obtained. We concluded that there could be multiple pathways of conformational folding which influence oxidative folding.
17
Zhang, Na. "Folding Analysis of Reduced Bovine Pancreatic Trypsin Inhibitor (BPTI) with Aromatic Thiols and Disulfides In Vitro". FIU Digital Commons, 2018. https://digitalcommons.fiu.edu/etd/3903.
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Almost all therapeutic proteins contain disulfide bonds to stabilize their native structure. Recombinant DNA technology enables many therapeutic proteins to be produced in bacteria, but the expression of native proteins is not always efficient due to the limited ability of bacteria to form disulfide bonds in vivo. It is often necessary to employ in vitro oxidative folding process to form the native disulfide bonds to obtain the native structure of disulfide-containing proteins. Aromatic disulfides are small molecules designed to match some of the physical properties of the active site of protein disulfide isomerase (PDI), which catalyzes the folding process of disulfide-containing proteins in eukaryotes.
Three aromatic thiols with varying charges, PA, SA and QAS thiol, were used to fold reduced BPTI in vitro. Bovine pancreatic trypsin inhibitor (BPTI) is positively charged (pI = 10.5) at pH 7.3, and we hypothesized that mixed disulfide intermediates formed between BPTI and negatively charged small molecule thiols were more likely to precipitate due to their minimized net charge. Protein precipitation was observed during folding with negatively charged thiols, PA and SA, but not positively charged thiol QAS. At the folding pH of 7.3, almost 90% of native BPTI was produced in 2 h with the conditions of 0.25 mM QAS disulfide and 10 mM QAS thiol. Only 25% of native BPTI was produced in 2 h with the best conditions for glutathione and glutathione disulfide. Aromatic thiols with an elongated alkyl group on the aromatic ring, butyl, hexyl and octyl thiol, were hypothesized to increased interactions with the hydrophobic core of disulfide-containing proteins during folding, allowing more facile access to buried disulfide bonds. However, the longer the hydrocarbon chain, the more likely protein precipitation was to occur. About 90% native BPTI was formed in 1 h with 0.25 mM hexyl disulfide and 10 mM hexyl thiol. A method using capillary electrophoresis (CE) to analysis the oxidative folding process of reduced BPTI with small molecule thiols and disulfides was also developed. Folding of reduced BPTI with QAS disulfide was analyzed using CE in a shorter run time. The consumption of protein samples and solvent solutions was minimized.
18
Evans, Steven John. "Structure, function and mechanism of action of bovine pancreatic deoxyribonuclease I : role of amino acid residues involved in phosphate contacts". Thesis, University of Newcastle Upon Tyne, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321857.
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Lequin, Olivier. "Etude par resonance magnetique nucleaire des angiogenines humaine et bovine, deux membres de la superfamille de la ribonuclease a pancreatique, impliques dans la neoformation des vaisseaux sanguins". Palaiseau, Ecole polytechnique, 1997. http://www.theses.fr/1997EPXX0036.
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Les angiogenines sont des proteines de 14 kda appartenant a la superfamille de la ribonuclease a pancreatique. A la difference des autres membres de la superfamille, elles ont une activite ribonucleasique extremement reduite, de specificite differente, et elles presentent la particularite d'induire la neoformation de vaisseaux sanguins. L'angiogenine bovine, purifiee a partir de lait de vache, a ete etudiee par des experiences homonucleaires exclusivement tandis que l'angiogenine humaine, surproduite dans la bacterie escherichia coli et marquee, a pu etre etudiee par rmn du proton et de l'azote #1#5n. Les structures de l'angiogenine humaine (bovine) ont ete determinees a partir de 1441 (1037) contraintes de distance, 79 (0) contraintes d'angle diedre et 100 (80) contraintes de liaison hydrogene. La precision obtenue pour les atomes du squelette est de 0,67 angstroms pour l'angiogenine humaine et 1,49 pour l'angiogenine humaine et 1,49 pour l'angiogenine bovine. Bien que la topologie globale des angiogenines soit similaire a celle de la ribonuclease a et d'autres proteines apparentees, des differences importantes ont ete observees, au niveau de l'extremite c-terminale et de plusieurs boucles, qui ont des repercussions sur l'arrangement des principaux sites fonctionnels, le site actif ribonucleasique et le site de liaison a un recepteur cellulaire. Le site de liaison de la partie pyrimidique du substrat (site b1) est obstrue par la chaine laterale du residu gln 117 (angiogenine humaine) ou glu 118 (angiogenine bovine), expliquant la tres faible activite ribonucleasique. Bien que le residu bloquant le site b1 semble assez blexible en solution, aucune conformation alternative ouverte de ce site n'a pu etre observee. Les structures sont proches des structures cristallographiques determinees par ailleurs. La rmn a permis d'apporter des informations supplementaires sur le mecanisme reactionnel, la dynamique et la protonation de certains residus du site actif ribonucleasique.
20
Sharma, Yugal K. "NMR Dynamic Characterization of a Disordered Peptide Derived From the V3 Loop of HIV-1 Both Free and Conjugated With Bovine Pancreatic Trypsin Inhibitor". University of Cincinnati / OhioLINK, 2000. http://rave.ohiolink.edu/etdc/view?acc_num=ucin971798331.
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Panse, Vikram G. "Interaction Of Chaperone SecB With Protein Substrates: A Biophysical Study". Thesis, Indian Institute of Science, 2000. http://hdl.handle.net/2005/242.
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In the cell, as in in vitro, the final conformation of a protein is determined by it's amino acid sequence (1). Some isolated proteins can be denatured and refolded in vitro in absence of extrinsic factors. However, in order to fold in the cell, the newly synthesized polypeptide chain has to negotiate an environment far more complex than that faced by the unfolded chain in vitro. Cells have evolved proteins called “chaperones” to assist folding and assembly of polypeptides (2). Thus, the linear sequence of a protein not only contains information that specifies the final three-dimensional functional form, but also recognition motifs, which can be recognized by the cellular folding machinery.
The work reported in this thesis is aimed at understanding some aspects of recognition of target substrates by the cytosolic chaperone, SecB, which forms part of the protein translocation machinery in E. coli. The sec pathway is involved in both translocation of precursor proteins across and the insertion of integral membrane proteins into the cytoplasmic membrane (3).
Chapter one discusses some general aspects of protein folding and briefly describes chaperone systems, which have been extensively characterized in literature.
Chapter two discusses the effect of chaperone SecB on the refolding pathway of a model substrate protein barstar, whose folding pathway has been extensively characterized (4,5). The effect of SecB on the refolding kinetics of the small protein barstar (wild type) and
fluorescein labeled C82A (single Cys mutant) in 1 M guanidine hydrochloride at pH 7.0 at 25 °C has been investigated using fluorescence spectroscopy. We show that SecB does not bind either the native or the unfolded states of barstar but binds to late near-native intermediate (s) along the folding pathway. ESR studies and fluorescence anisotropy measurements show that SecB forms stable complexes with the near-native intermediate (s). For barstar, polypeptide collapse and formation of a hydrophobic surface are required for binding to SecB. Steady state polarization measurements indicated the presence of stable complexes of barstar bound to SecB. Studies on the spin labeled C82A show an immobilization of the spin label adduct at the 40th position of barstar, suggesting that the binding of SecB to barstar occurs in that region. SecB does not change the apparent rate constant of barstar refolding. The kinetic data for SecB binding to barstar are not consistent with simple kinetic partitioning models (6).
Chapter three discusses the energetics of substrate:SecB interactions using the following model protein substrates: unfolded RNase A, BPTI, partially folded disulfide intermediates of alpha-lactalbumin,. The thermodynamics of binding of unfolded polypeptides to the chaperone SecB were investigated in vitro by isothermal titration calorimetry and fluorescence spectroscopy. The heat capacity changes observed on binding the reduced and carboxamidomethylated forms of alpha-lactalbumin, BPTI, and RNase A were found to be -0.10, -0.29 and -0.41 kcal mol-1 K-1 respectively and suggest that between 7 and 29 residues are buried upon substrate binding to SecB. In all cases binding occurs with a stoichiometry of one polypeptide chain per monomer of SecB. The data are consistent with a model where SecB binds substrate molecules at an exposed hydrophobic cleft (7).
Chapter four discusses the thermodynamics of unfolding to gain insights into the mechanism of assembly and stability of the tetrameric structure. The thermodynamics of unfolding of SecB was studied as a function of protein concentration, by using high sensitivity-differential scanning calorimetry and spectroscopic methods. The thermal unfolding of tetrameric SecB is reversible and can be well described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers. The
value of ACP obtained was 10.7 ± 0.7 kcal mol-1 K-1, which is amongst the highest measured for a multimeric protein. At 298 K, pH 7.4. the AG°U for the SecB tetramer is 27.9 ± 2 kcal mol-1. Denaturant mediated unfolding of SecB was found to be irreversible. The reactivity of the 4 solvent exposed free thiols in tetrameric SecB is salt dependent. The kinetics of reactivity suggests that these four Cysteines are in close proximity to each other and that these residues on each monomer are in chemically identical environments. The thermodynamic data suggest that SecB is a stable, well folded and tightly packed tetramer and that substrate binding occurs at a surface site rather than at an interior cavity (8).
Chapter five discusses the bound state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI), as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods. BPTI is a 58 residue protein and contains 3 disulfide groups between residues 5 and 55, 14 and 38, and 30 and 51. Single disulfide mutants of BPTI were reduced and the free cysteines were labeled with either thiol-specific spin labels or pyrene maleimide. The relative proximity of labeled residues was studied using either electron spin resonance spectroscopy or fluorescence spectroscopy. The data suggest that SecB binds a collapsed coil of reduced unfolded BPTI, which then undergoes a structural rearrangement to a more extended state upon binding to SecB. Binding occurs at multiple sites on the substrate and the binding site on each SecB monomer accommodates less than 21 substrate residues. In addition, we have labeled four, solvent accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein. The ESR data suggest that these cysteine residues are in close proximity when no substrate protein is bound, but move away from each other when SecB binds substrate. This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein.
Chapter six discusses the mechanism of dissaggregation of a model peptide aggregate by chaperone SecB. The Hspl04, Hsp70 and Hsp40 chaperone system are capable of dissociating aggregated state(s) of substrate proteins, though little is known of the
mechanism of the process. The interaction of the B chain of insulin with chaperone SecB was investigated using light scattering, pyrene excimer fluorescence and electron spin resonance spectroscopy. We show that SecB prevents aggregation of the B chain of insulin. We show that SecB is capable of dissociating soluble B chain aggregate as monitored by pyrene fluorescence spectroscopy. The kinetics of dissociation of the B chain aggregate by SecB has also been investigated to understand the mechanism of dissociation. The data suggests that SecB does not act as a catalyst in dissociation of the aggregate to individual B chains, rather it binds the small population of free B chains with high affinity, thereby shifting the equilibrium from the ensemble of the aggregate towards the individual B chains. Thus SecB can rescue aggregated, partially folded /misfolded states of target proteins by a thermodynamic coupling mechanism when the free energy of binding to SecB is greater than the stability of the aggregate. Pyrene excimer fluorescence and ESR methods have been used to gain insights on the bound state conformation of the B chain to chaperone SecB. The data suggests that the B chain is bound to SecB in a flexible extended state in a hydrophobic cleft on SecB and that the binding site accommodates approximately 10 residues of substrate (9).
22
Ribó, i. Panosa Marc. "Purificació, caracterització i clonatge de la ribonucleasa de pàncreas humà". Doctoral thesis, Universitat de Girona, 1994. http://hdl.handle.net/10803/96757.
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The answer to whether or not, human pancreatic ribonuclease may have a diagnostic value as a serum marker of pancreatic disease, depend upon the specific detection of ribonucleases originated in pancreas amogn those coexisting in serum. From our point of view, two approaches would be necessary. On one hand the characterization of the carbohydrate component of the human pancreatic ribonuclease because glycosylation is organ-specific. On the other hand, obtain monoclonal antibodies in order to specifically recognize the secretory -typeribonuclease in serum. The purification system that has been established consists of two chromatographic steps using HPLC. The first, an exchange cromatography, allows for a partial purification of ribonuclease component. The second, a reversed-phase chromatography, resolves chromatographically the human pancreatic ribonuclease heterogeneity into different fractionsLa resposta a si la ribonucleasa de pàncreas humà pot tenir algun valor diagnòstic com a marcador sèric de disfuncions pancreàtiques passa per la identificació de forma inequívoca de la ribonucleasa de pàncreas humà en sèrum. Segons el nostre enfoc, caldrien dues aproximacions, d’una banda la caracterització a nivell glucídic de la ribonucleasa de pàncreas humà, i de l’altra, el desenvolupament d’anticossos monoclonals dirigits a discriminar la ribonucleasa de tipus secretori d’entre els diferents tipus que coexisteixen en sèrum. En aquest sentit s’ha establert un sistema de purificació de la ribonucleasa pancreàtica humana que fos ràpid i repetitiu que respectés l’heterogeneïtat glucídica per a la seva posterior caracterització. El sistema de purificació establert a partir de pàncrees obtinguts a partir de donants sans d’òrgans consta de dues etapes cromatogràfiques per HPLC. La primera, una cromatografia de bescanvi catiònic, possibilita una purificació parcial, mentre la cromatografia de la fase inversa permet resoldre cromatogràficament l’heterogeneïtat de la ribonucleasa humana de pàncreas en diferents fraccions
23
Poi, Ming. "Low-barrier hydrogen bonding in bovine pancreatic Phospholipase A₂ and somatic INK4A-ARF locus mutations : a significant mechanism of gene inactivation in head and neck squamous cell carcinomas /". The Ohio State University, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=osu1486402544589967.
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Haimann, Michaela Maria [Verfasser] y Wolfgang E. [Akademischer Betreuer] Trommer. "Proteintransport in E. coli: Untersuchung der Konformationsänderungen des Chaperons SecB durch Bindung an das Modellsubstrat Bovine Pancreatic Trypsin Inhibitor (BPTI) mit Hilfe der EPR-Spektroskopie / Michaela Haimann. Betreuer: Wolfgang E. Trommer". Kaiserslautern : Universitätsbibliothek Kaiserslautern, 2012. http://d-nb.info/1021574260/34.
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Zhu, Hongxin. "Structure-Function Studies of Bovine Pancreatic Phospholipase A2: The Roles of N-Terminal Residues in The Interfacial Activation and The Roles of Disulfide Bonds in The Structure, Stability, and Catalytic Function Cloning, Expression, Purification... /". The Ohio State University, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487931993466424.
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Marty, Jean-Louis. "Métabolisation des phenylcarbamates herbicides : rôle des enzymes et des microorganismes". Perpignan, 1987. http://www.theses.fr/1987PERP0039.
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Bosch, i. Grau Montserrat. "Producció i caracterització de variants de la ribonucleasa pancreàtica humana dissenyades per a adquirir propietats citotòxiques". Doctoral thesis, Universitat de Girona, 2003. http://hdl.handle.net/10803/7612.
Texto completoResumen
Amb la finalitat d'aprofundir en les bases moleculars de la citotoxicitat de les ribonucleases pancreàtiques, es van construir variants derivades de l'HP-RNasa seguint dues estratègies. En la primera, es van generar variants de l'enzim resistents a l'acció de l'inhibidor proteic de les ribonucleases (hRI), substituint residus implicats en la interfície de contacte entre la ribonucleasa i l'hRI. En la segona, es va addicionar el motiu RGD en regions de superfície de la proteïna implicades en la formació del complex amb l'hRI, a fi de promoure la seva interacció amb la membrana plasmàtica de les cèl·lules i a la vegada disminuir l'afinitat de les variants per l'hRI. Es va comprovar que només les variants portadores de substitucions múltiples adquirien la capacitat de resistència a l'hRI.L'estudi del percentatge d'inhibició de la síntesi proteica en cèl·lules incubades amb cadascuna de les variants va mostrar que només dues de les variants construïdes havien adquirit propietats citotòxiques. La citotoxicitat més elevada la va presentar una variant que no era resistent a l'hRI, amb valors que eren només entre 5 i 15 vegades inferiors als de l'onconasa. Aquest resultat demostrà que la sensibilitat a l'hRI no és necessàriament un paràmetre limitant per a la citotoxicitat de les ribonucleases. Cap de les variants que incorporava un motiu RGD presentà citotoxicitat, evidenciant que aquest motiu no és efectiu a fi de dotar les ribonucleases pancreàtiques de propietats citotòxiques.
Es van estudiar les bases moleculars de la citotoxicitat de la variant més citotòxica. En primer lloc, l'anàlisi de la internalització per marcatge radioactiu d'aquesta variant en relació amb l'onconasa i amb altres variants de l'HP-RNasa no citotòxiques, va posar en evidència que només l'onconasa era internalitzada eficientment. Es descartava així la possibilitat que l'acció citotòxica de l'enzim estudiat fos conseqüència d'una major eficiència d'endocitosi. També es va comprovar que l'addició del motiu RGD no era capaç de promoure la internalització de les proteïnes amb més eficàcia. Per microscòpia confocal de fluorescència, les variants humanes només es van començar a detectar a l'interior de la cèl·lula a partir de les 24 h d'incubació.
Totes les variants generades van presentar una eficiència catalítica superior al 50 % de l'activitat de la seva proteïna parental, PM5, indicant que probablement l'estructura del centre actiu no havia estat afectada de manera dràstica per les substitucions introduïdes. No obstant, en tots els casos es va produir una disminució en la termoestabilitat respecte a PM5. Aquest resultat indicà que la correlació descrita a la bibliografia entre l'increment de termoestabilitat i l'increment de citotoxicitat per les ribonucleases no sempre es compleix.
Per microscòpia confocal es va comprovar que tant la proteïna més citotòxica, com una variant no citotòxica resistent a l'hRI, així com la proteïna parental, seguien la via de degradació lisosomal. Aquesta ruta de trànsit no va ser afectada per l'addició de drogues que alteren les vies de trànsit retrògrad (monensina i brefeldina A), però sí per l'addició de la bafilomicina A1, una droga que neutralitza el pH endosomal i que va actuar alentint el trànsit de les proteïnes als lisosomes. D'acord amb aquests resultats, els valors de citotoxicitat de les variants es van incrementar de manera significativa només en presència de bafilomicina A1, suggerint que les ribonucleases transloquen al citoplasma a partir d'algun punt de la via de trànsit endosomal.
Es va comprovar que l'acció de la variant més citotòxica era deguda a que l'addició d'un segon motiu de tres Arg en PE5 dota a aquesta proteïna amb un senyal de transport nuclear. La fracció d'enzim que aconsegueix translocar al citoplasma a partir d'algun punt de la via endosomal previ als lisosomes, és conduït ràpidament al nucli de la cèl·lula per mitjà del mecanisme clàssic de transport actiu. Per la seva afinitat amb l'rRNA, l'enzim es concentra en el nuclèol, on probablement duu a terme la seva activitat catalítica. La interacció d'aquesta variant amb els receptors nucleocitoplasmàtics, les importines, impediria per altra banda el bloqueig de l'enzim per part de l'hRI.
Els resultats obtinguts presenten una nova estratègia de disseny de ribonucleases citotòxiques, basada en l'addició de segments NLS a fi de promoure el transport nuclear dels enzims. Aquesta estratègia podria permetre superar limitacions que fins al moment han estat descrites com a limitants de la citotoxicitat de les ribonucleases pancreàtiques, com la sensibilitat a l'hRI o la baixa eficiència d'internalització.
The main objective of this thesis is to study the molecular bases of the cytotoxicity of certain ribonucleases. With the final aim to obtain cytotoxic variants derived from human pancreatic ribonuclease. For this purpose, we created variants derived from HP-RNase by using two different strategies. In the first, variants of the enzyme that were resistant to the action of the protein inhibitor of the ribonuclease (RI) were generated, replacing residues involved in the contact interfase between the ribonuclease and RI. In the second, RGD motifs were added to the surface of the proteins involved in the formation of the RI complex, with the aim of promoting their interaction with the cell plasmatic membrane, whilst at the same time decreasing the variant's affinity for RI. We showed that only variants carrying multiple substitutions acquired the capacity to resist the RI.
The study of the percentage of inhibition of protein synthesis in incubated cells using each of the variants showed that only two variants had acquired cytotoxic properties. The highest level of cytotoxicity found in a non-resistant variant to RI had a value that was only 5 and 25 times lower than those registered by Onconase®. This result shows that RI sensitivity is not a limiting factor for the cytotoxicity of the ribonuclease. None of the variants which contained RGD motifs showed any sign of toxicity, suggesting that for this reason it is not effective in giving pancreatic ribonuclease cytotoxic properties.
The cytotoxic molecular bases of the most cytotoxic variant were studied. Firstly by the analysis of the internalisation of this particular variant by radioactive marking in relation to Onconase and other non-cytotoxic variants of HP-RNase, which showed that only Onconase was effectively internalised. Thus the possibility that the cytotoxic action of the enzyme under observation was a result of a more efficient endocytosis was ruled out. It was also shown that the addition of the RGD motif was unable to encourage the internalisation of the proteins more effectively. Using confocal microscopy, the human variants only began to be noted inside the cell after 24 hours incubation.
All the variants that were created retained a catalytic efficiency that was never less than 50 % of the catalytic activity achieved by the parent protein PM5. This suggests that the structure within the active centre had not been affected in any serious way by the introduction of the substitutions. However a decrease in thermostabilty was noted across the board with regards to PM5. This result indicates that the correlation mentioned in the bibliography between the increase of thermostability and the increase of cytotoxicity of the ribonuclease does not always exist.
Using a confocal microscope, we confirmed that both the most cytotoxic protein, such as a non-cytotoxic variant resistant to RI, and the parent protein, followed the same lysosomal pathway of degradation. This outcome was unaffected by the addition of drugs which can change retrograde transit pathways (monensine and brefeldine A), but was effected by the addition of bafilomicine A1 a drug which neutralises endosomal pH and which in this case acted by slowing down the movement of proteins to lysosomes. In accordance with these results, the cytotoxicity values of the variants were significantly increased only by the presence bafilomicine A1 suggesting that the ribonuclease translocate into the cytoplasm starting from a point somewhere along the endosomal transit pathway.
We confirmed that the behaviour of the most cytotoxic variant was due to the fact that the addition of a second motif of 3 Arg in PE5 endowed the protein with a nuclear transport signal. The division of the enzyme that translocates into the cytoplasm (from somewhere along the endosomal transit path before the lysosomes) is rapidly moved towards the core of the cell via the conventional mechanism for nucleus transport. Due to its affinity for rRNA, the enzyme gathers in the nucleolus, where it probably carries out its catalytic activity. On the other hand, the interaction of this variant with nucleocytoplasmatic receptors will prevent the RI from inhibiting the enzyme.
These results offer a new strategy for the design of cytotoxic ribonuclease, based on the addition of NLS motifs, with the aim of encouraging the nuclear transport of enzymes. This strategy could allow one to overcome limitations that up until now have been the down-side to the cytotoxicity of pancreatic ribonuclease, such as a sensitivity for RI or the limited efficiency of internalisation.
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Rodríguez, Maynou Montserrat. "Characterization of cytotoxic ribonucleases: from the internalization pathway to the importance of dimeric structures". Doctoral thesis, Universitat de Girona, 2006. http://hdl.handle.net/10803/7622.
Texto completoResumen
En aquesta tesi s'ha caracteritzat la ruta d'internalització de l'onconasa, una RNasa citotòxica. Els resultats indiquen que l'onconasa entra a les cèl·lules per la via dependent de clatrina i del complex AP-2. Seguidament es dirigeix als endosomes de reciclatge i es a través d'aquesta ruta que la proteïna exerceix la citotoxicitat. Per altra banda, els resultats d'aquest treball demostren que PE5, una variant citotòxica de la ribonucleasa pancreàtica humana (HP-RNasa), interacciona amb la importina mitjançant diferents residus que tot i que no són seqüencials, es troben propers en l'estructura tridimensional d'aquesta proteïna. PM8 és una HP-RNasa amb estructura cristal·logràfica dimèrica constituïda per intercanvi de dominis N-terminals. En aquesta tesi s'han establert les condicions per estabilitzar aquest dimer en solució i també es proposa un mecanisme per la dimerització.In this thesis it has been characterized the internalization pathway of onconase, which is a cytotoxic ribonuclease. The results show that onconase enters cells using AP-2/clathrin mediated pathway and then is routed to the recycling endosomes. In addition, the results show that this is the route used by onconase to perform its cytotoxicity. On the other hand, the results indicate that PE5, a cytotoxic human pancreatic ribonuclease (HP-RNase), interacts with importin α using different residues that although they are scattered along the sequence, they are close in the three-dimensional structure of the protein. PM8 constitutes a crystallographic dimer by the exchange of the N-terminal domains. In this thesis it has been investigated the solution conditions that favour the dimeric form and it is proposed a dimerization process of this variant. Finally, the pattern of substrate cleavage is studied by HP-RNase.
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Vert, Company Anna. "Molecular mechanism of PE5-induced cytotoxicity and generation of new cytotoxic nuclear-directed". Doctoral thesis, Universitat de Girona, 2014. http://hdl.handle.net/10803/283575.
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Cytotoxic ribonucleases are promising agents to be used in the treatment of cancer. Our group previously described a cytotoxic human pancreatic ribonuclease variant, named PE5, which carries a nuclear localization signal. This protein is routed into the nucleus, where it cleaves nuclear RNA inducing the apoptosis of cancer cells. In the present work, the molecular mechanism of PE5-induced cytotoxicity has been investigated using global gene expression and miRNA microarrays. The results indicate that this ribonuclease causes pleiotropic effects and regulates the expression of numerous genes and miRNAs. On the other hand, we have improved the antitumor properties of PE5. First, we have obtained PE10, which is as cytotoxic as PE5 but it is potentially less immunogenic because its sequence is more similar to that of the wild type human pancreatic ribonuclease. And second, we have obtained NLSPE5, which exhibits 6-14 times higher cytotoxicity for tumor cells than PE5Les ribonucleases citotòxiques són proteïnes amb un gran potencial per ser utilitzades en el tractament del càncer. El nostre grup va descriure una variant citotòxica de la ribonucleasa pancreàtica humana, anomenada PE5, que incorpora un senyal de localització nuclear. Aquesta proteïna es dirigeix al nucli, on degrada RNA nuclear induint així l’apoptosi de les cèl•lules tumorals. En aquest treball s’ha investigat el mecanisme de citotoxicitat de PE5 utilitzant microarrays globals d’expressió gènica i de miRNAs. Els resultats obtinguts indiquen que PE5 causa efectes pleiotròpics i regula l’expressió de nombrosos gens i miRNAs. D’altra banda, s’han millorat les propietats antitumorals de PE5. Primer, s’ha produït PE10, la qual presenta la mateixa citotoxicitat que PE5 però és potencialment menys immunogènica perquè la seva seqüència és més similar a la de la ribonucleasa pancreàtica humana. En segon lloc, s’ha construït NLSPE5, la quals exhibeix una citotoxicitat 6-14 vegades superior a PE5
30
Robert, Stéphane. "Acylation de protéines en micelles inverses". Compiègne, 1995. http://www.theses.fr/1995COMP836S.
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Dans le but de greffer une ou deux chaînes d'acides gras à des protéines hydrophiles afin de leur conférer une capacité d'interaction avec les membranes (et plus largement d'étudier le phénomène de l'acylation naturelle des protéines), un système micellaire de surfactant dans un solvant organique apolaire a été utilisé comme milieu de réaction pour la modification protéique par des réactifs lipophiles comme les chlorures d'acides gras. Le degré d'hydratation du surfactant qui gouverne la taille des micelles inverses, et d'autres paramètres tels que le pH du compartiment aqueux, la quantité de protéine micellisée et le ratio molaire réactif/protéine ont été étudiés et optimisés pour l'acylation de la ribonucléase A avec le chlorure d'acide myristique, puis appliqués pour les chlorures d'acides caprylique, palmitique, palmitoléique et stéarique. Une méthode de séparation utilisant la chromatographie liquide haute performance en phase inverse permet de récupérer en deux fractions distinctes la protéine non modifiée et la protéine modifiée. Les quantités récoltées sont de l'ordre de la dizaine de milligrammes et peuvent être augmentées. L'électrophorèse capillaire de ces échantillons a confirmé leur homogénéité et leur pureté. Les résultats obtenus par spectrométrie de masse (dispersion par électrospray) sur les dérivés acyles de la ribonucléase A ont permis de certifier qu'une seule chaîne acyl est fixée par monomère. Un séquençage par dégradation d'Edman a montré une fixation amino-terminale des résidus d'acides gras sur la lysine 1 de la ribonucléase A. Ces deux fractions conservent leur activité cataytique. La stabilité thermique des dérivés protéiques acyles est même augmentée par rapport à l'enzyme native, comme le montrent les études microcalorimétriques. En conclusion, nous avons réalisé une acylation amino-terminale en conservant l'intégrité biologique de la ribonucléase A
31
Thompson, James E. "Catalysis by bovine pancreatic ribonuclease A - energetics and contributions from the active-site histidines". 1995. http://catalog.hathitrust.org/api/volumes/oclc/34764021.html.
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Chang, Yung-Ming y 張永明. "Bovine pancreatic Deoxyribonuclease F". Thesis, 1993. http://ndltd.ncl.edu.tw/handle/66508029209635905830.
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WANG, ZHENG-QING y 王政清. "Hydrolysis of synthetic oligodeoxyribonucleatides catalyzed by bovine pancreatic phosphodiesterase". Thesis, 1991. http://ndltd.ncl.edu.tw/handle/27932626382131757973.
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何恒堅. "The Structure and function of bovine pancreatic deoxyribonuclease I". Thesis, 1991. http://ndltd.ncl.edu.tw/handle/60908482001114226076.
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李宜軒. "= Purification and study of bovine pancreatic deoxyribonuclease I cysteine mutants". Thesis, 2002. http://ndltd.ncl.edu.tw/handle/29895655039670564118.
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VOTTARIELLO, FRANCESCA. "OLIGOMERIZATION OF RNase A:a) A STUDY OF THE INFLUENCE OF SERINE 80 RESIDUE ON THE 3D DOMAIN SWAPPING MECHANISMb) “ZERO-LENGTH” DIMERS OF RNase A AND THEIR CATIONIZATION WITH PEI". Doctoral thesis, 2010. http://hdl.handle.net/11562/344075.
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"Zero-length" dimers of ribonuclease A, a novel type of dimers formed by two RNase A molecules bound to each other through a zero-length amide bond [Simons, B.L. et al. (2007) Proteins 66, 183-195], were analyzed, and tested for their possible in vitro cytotoxic activity. Results: (i) Besides dimers, also trimers and higher oligomers can be identified among the products of the covalently linking reaction. (ii) The "zero-length" dimers prepared by us appear not to be a unique species, as was instead reported by Simons et al. The product is heterogeneous, as shown by the involvement in the amide bond of amino and carboxyl groups others than only those belonging to Lys66 and Glu9. This is demonstrated by results obtained with two RNase A mutants, E9A and K66A. (iii) The "zero-length" dimers degrade poly(A).poly(U) (dsRNA) and yeast RNA (ssRNA): while the activity against poly(A).poly(U) increases with the increase of the oligomer's basicity, the activity towards yeast RNA decreases with the increase of oligomers' basicity, in agreement with many previous data, but in contrast with the results reported by Simons et al. (iv) No cytotoxicity against various tumor cells lines could be evidenced in RNase A "zero-length" dimers. (v) They instead become cytotoxic if cationized by conjugation with polyethylenimine [Futami, J. et al. (2005) J. Biosci. Bioengin. 99, 95-103]. However, polyethylenimine derivatives of RNase A "zero-length" dimers and native, monomeric RNase A are equally cytotoxic. In other words, protein "dimericity" does not play any role in this case. Moreover, (vi) cytotoxicity seems not to be specific for tumor cells: polyethylenimine-cationized native RNase A is also cytotoxic towards human monocytes."Zero-length" dimers of ribonuclease A, a novel type of dimers formed by two RNase A molecules bound to each other through a zero-length amide bond [Simons, B.L. et al. (2007) Proteins 66, 183-195], were analyzed, and tested for their possible in vitro cytotoxic activity. Results: (i) Besides dimers, also trimers and higher oligomers can be identified among the products of the covalently linking reaction. (ii) The "zero-length" dimers prepared by us appear not to be a unique species, as was instead reported by Simons et al. The product is heterogeneous, as shown by the involvement in the amide bond of amino and carboxyl groups others than only those belonging to Lys66 and Glu9. This is demonstrated by results obtained with two RNase A mutants, E9A and K66A. (iii) The "zero-length" dimers degrade poly(A).poly(U) (dsRNA) and yeast RNA (ssRNA): while the activity against poly(A).poly(U) increases with the increase of the oligomer's basicity, the activity towards yeast RNA decreases with the increase of oligomers' basicity, in agreement with many previous data, but in contrast with the results reported by Simons et al. (iv) No cytotoxicity against various tumor cells lines could be evidenced in RNase A "zero-length" dimers. (v) They instead become cytotoxic if cationized by conjugation with polyethylenimine [Futami, J. et al. (2005) J. Biosci. Bioengin. 99, 95-103]. However, polyethylenimine derivatives of RNase A "zero-length" dimers and native, monomeric RNase A are equally cytotoxic. In other words, protein "dimericity" does not play any role in this case. Moreover, (vi) cytotoxicity seems not to be specific for tumor cells: polyethylenimine-cationized native RNase A is also cytotoxic towards human monocytes.
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Gao, Qian Wen y 高千雯. "Investigation of the histidine residues in bovine pancreatic DNase I by chemical modification". Thesis, 1996. http://ndltd.ncl.edu.tw/handle/90899859841354085914.
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Kanaujia, Shankar Prasad. "Structural Studies On Bovine Pancreatic Phospholipase A2 And Proteins Involved In Molybdenum Cofactor Biosynthesis". Thesis, 2010. http://etd.iisc.ernet.in/handle/2005/2000.
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We have carried out structural studies on bovine pancreatic phospholipase A2 (BPLA2) and two proteins involved in molybdenum cofactor (Moco) biosynthesis pathway. In addition, molecular-dynamics simulations and other analyses have been performed to corroborate the findings obtained from the crystal structures.
Crystal structures of the three active-site mutants (H48N, D49N and D49K) of BPLA2 were determined to understand the mechanism by which the mutant H48N is able to catalyze the reaction of phospholipid hydrolysis and to see the effect of the loss of Ca 2+ ion in the active site of D49N and D49K mutants. We found that Asp49 could possibly play the role of a general base instead of His48 in the case of the H48N mutant. In the case of D49N and D49K mutants, the active site of the enzyme is perturbed, whereas the overall tertiary structure of these mutants is intact. In addition, a total of 24 invariant water molecules were identified in all of the crystal structures of BPLA2 available in its archive, PDB. Out of these, four water molecules are essential for the catalytic activity, whereas, the remaining water molecules play a role in the stability of the enzyme.
In addition, structural studies on two proteins MoaC and MogA involved in Moco biosynthesis pathway have been carried out. For the first time, crystal structure of MoaC bound with GTP molecule has been reported. The gene id TTHA0341, which is mentioned as MoaB in the CMR database, was annotated as MogA based the comparative analysis of sequences and structures (with the present work and the structures available in the literature). The role of N-and C-termini of MoaB and MogA proteins were proposed that these residues might stabilize the substrate and/or product molecule in the active site. In addition, the residues involved in the oligomerization are compared with MD simulations. The molecular docking studies show that MoaB proteins show more preference to GTP than ATP. The comparison of the two active (MPT and AMP-binding) sites revealed that MPT-binding site is preferred over AMP-binding site for nucleotide binding.
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Ching-Ying, Chen y 陳靜瑩. "Bovine pancreatic deoxyribonuclease：cDNA cloning and the functional roles of the two structural calcium sites". Thesis, 2001. http://ndltd.ncl.edu.tw/handle/98905903654824770635.
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博士國立臺灣大學
生化學研究所
89
The bovine pancreatic (bp) DNase gene was cloned from bp cDNA and expressed in E. coil. A polynucleotide sequence of 1295-base was deduced from clones of the cDNA. The sequence showed an open reading frame, which can be translated as a 282-amino acid polypeptide, including a hydrophobic signal peptide and the polypeptide of bpDNase. An expression plasmid was constructed by inserting into the vector pET15b a cDNA fragment coding for bpDNase ligated with a hexanucleotide coding for Met-Ala at the 5'-end. The plasmid was transformed into E. coli strain BL21(DE3)pLysE and the active bovine recombinant DNase (brDNase) was produced after induction of protein synthesis. From the induced culture medium, brDNase was purified with a Mono Q column. The purified brDNase shows a molecular mass of 29 kDa and has the same specific activity as does bpDNase. The NH2-terminus of brDNase is Ala, not Met, and at position 19, corresponding to the carbohydrate attachment site of bpDNase, Asn18 is non-glycosylated. Using site-directed mutagenesis, we changed the brDNase active site His134 to Gln. The brDNase(H134Q), like bpDNase, was purified on a Mono Q column based on the principle of calcium affinity elution, indicating the retention of the two Ca2+-binding sites. The mutant protein thus obtained was not active. The two amino acid residues, Asp99 and Asp201, which involved in the coordination of the two calcium atoms found in the X-ray structure of bpDNase, were individually changed by site-directed mutagenesis. The two altered proteins, brDNase(D99A) and brDNase(D201A), expressed in E. coli, were purified by Ca2+-affinity chromatography. Equilibrium dialysis showed that mutation destroyed one Ca2+-binding site each in brDNase(D99A) and brDNase(D201A). Compared to bpDNase, when the large molecular DNA was used as substrate, the Vmax value for brDNase(D99A) remained unchanged and that for brDNase(D201A) was decreased, while the Km values for the two variants were increased 2-3 fold. When the small molecular NPPP was used as substrate, the brDNase(D99A) had 33 % of the bpDNase activity remaining and the brDNase(D201A), 25 %. Restriction enzymes were used to cut pETD99A and pETD201A and the products were used to construct a double mutant (D99A/D201A) plasmid, pETDM. This plasmid expressed a brDNase (D99A/D201A) which had, based on Western blotting and activity staining, 1.2 % of the bpDNase activity remaining. Like bpDNase, brDNase(D99A) was able to make double-scission on duplex DNA with Mg2+ plus Ca2+ and was effectively protected by Ca2+ from the trypsin inactivation. But under the same conditions, brDNase(D201A) lost the double-scission ability and was not protected by Ca2+. Nevertheless, the two variant proteins retained the characteristics of the Ca2+-induced conformational changes and the Ca2+-protection against the β-mercaptoethanol disruption of the essential disulfide bond, suggesting that other weaker Ca2+-binding sites not found in the X-ray structure were responsible for these properties. Therefore, the two structural calcium atoms are not for maintaining the overall conformation of the active DNase, but rather play the role in the fine-tuning of the DNase activity.
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Lo, Ting y 駱亭. "The thioredoxin-like activity and calcium binding ability of the short-range disulfide in bovine pancreatic deoxyribonuclease". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/72072778748080749186.
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碩士國立臺灣大學
生物化學暨分子生物學研究所
93
Bovine pancreatic deoxyribonuclease (bpDNase) is the best-characterized DNase. It is composed of 260 amino acids, and is a glycoprotein with a molecular weight of 31 kDa. One large (C173-C209) and one small (C101-C104) disulfide loops were found in bpDNase. Earlier studies showed that the large disulfide loop was responsible for the enzyme conformation. When the large disulfide loop was reduced, bpDNase lost its enzyme activity. In contrast, reduction of the small loop resulted in an enzyme with the full activity. However, sequence alignment of DNases from various species revealed that this small loop was still highly conserved among species. Moreover, because the structure-based sequence alignment revealed homology between the small disulfide loop and the active site motif (-CXXC-) of thioredoxin, we are interested in seeking the possible biological roles of the small loop in bpDNase. Although not related to enzyme activity, the nonessential disulfide C101-C104 might be very important for other unknown functions. The importance of this disulfide also can be found in conservative sequences of various DNases. According to our recent studies, the reduced bpDNase actually contained the thioredoxin-like activity based on the rate of insulin precipitation assay. In order to gain further insight into the biological functions of the small loop, four double (E102G/S103P、E102P/S103G、G100K/G105W、G100W/G105K) and two quadruple (G100K/E102P/S103G/G105W、G100W/E102G/S103P/G105K) mutants were constructed using site-directed mutagenesis. Recombinant proteins were expressed in E. coli strain BL21(DE3)pLysE and were purified through a SOURCE 15Q anion and a S HyperD cation-exchange columns. SDS-PAGE with silver stain confirmed the homogeneity of the purified brDNase variants. Most of the recombinant proteins possess similar specific activities as the wild type bpDNase. However, quadruple mutant KPGW exhibited only half of the activity. CD spectra analysis also revealed significant different for this mutant. In our studies, we found that all these brDNase variants were able to accelerate the rate of insulin precipitation. And the highest thioredoxin-like activity (66%) of the quadruple mutant WGPK suggests that the conserved sequence (-WCGPCK-) of thioredoxin is crucial for its activity. Previous studies also showed that these two disulfide bonds were correlated with the two calcium binding sites of bpDNase, site I and site II. It was shown that the binding of calcium of site II is responsible for the conformation of the loose loop, C101-C104. We found that among brDNase variants which were mutated around the small loop only the reversed-sequence quadruple mutant KPGW presented a weaker binding ability, probably due to the alteration of its secondary structure.
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Chen, Wei-Jung y 陳威戎. "Biological Functions of the Disulfides in Bovine Pancreatic Deoxyribonuclease and the Involvement of its N- and C-Terminal Fragments in Active Protein Folding". Thesis, 2004. http://ndltd.ncl.edu.tw/handle/46620305879702105569.
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博士國立臺灣大學
生物化學暨分子生物學研究所
93
Bovine pancreatic deoxyribonuclease (bpDNase) is the best-characterized DNase which cleaves double stranded DNA with no sequence specificity. It was the first DNase discovered and sequenced with the conventional protein sequencing technique and the X-ray structure was resolved at 2.0 Å with refinement. In this dissertation, the biological functions of the disulfides in bpDNase and the involvement of its N- and C-terminal fragments in active protein folding were demonstrated. The biochemical functions of the small non-essential (C101-C104) and the large essential (C173-C209) disulfides in bpDNase have been characterized using alanine mutants [brDNase(C101A)] and [brDNase(C173A) and brDNase(C209A)], respectively. In addition, we have characterized the effects of an additional third disulfide [brDNase(F192C/A217C)]. Without the Ca2+ protection, bpDNase and brDNase(C101A) were readily inactivated by trypsin while brDNase(F192C/A217C) remained active. With Ca2+, all forms of DNase, except for brDNase(C101A), were protected against trypsin. All forms of DNase, after being dissolved in 6 M guanidine-HCl, were fully reactivated by diluting into a Ca2+-containing buffer. However, when diluted into a Ca2+-free buffer, bpDNase and brDNase(C101A) remained inactive, but 60% of the bpDNase activity was restored with brDNase(F192C/A217C). When heated, bpDNase was inactivated at a transition temperature of 65 �aC, brDNase(C101A) at 60 �aC, and brDNase(F192C/A217C) at 73 �aC, indicating that the small disulfide, albeit not essential for activity, is important for the structural integrity, and that the introduction of a third disulfide can further stabilize the enzyme. When pellets of brDNase(C173A) and brDNase(C209A) in inclusion bodies were dissolved in 6 M guanidine-HCl and then diluted into a Ca2+-containing buffer, 10-18% of the bpDNase activity was restored, suggesting that the “essential” disulfide is not absolutely crucial for enzymatic catalysis. Owing to the structure-based sequence alignment revealing homology between the “non-essential” disulfide of bpDNase and the active site motif of thioredoxin, we measured 39% of the thioredoxin-like activity for bpDNase based on the rate of insulin precipitation (�槎650nm/min). Thus, the disulfides in bpDNase not only play the role of stabilizing the protein molecule but also may engage in biological functions such as the disulfide/dithiol exchange reaction. The X-ray crystal structure of bpDNase revealed that its N- and C-termini were in close proximity forming an anti-parallel ��-sheet structure, and these terminal amino acid sequences were highly conserved among species. The involvement of this ��-sheet structure in the active protein folding of bpDNase was thus investigated via a series of deletion and substitution variants. Several substitution variants of the N-terminal Leu1 and C-terminal Leu259 were shown to be fully active, indicating that the main-chain hydrogen bonding, rather than the side-chain interactions, was crucial for the formation of the anti-parallel ��-sheet structure. The variant with only the C-terminal Thr260 deleted, remained active. However, the other deletion variants, in which 2 to 10 amino acid residues were removed from the C- or N-terminus, all lost the DNase activity. When synthetic peptides corresponding to the deleted N- and C-terminal sequences were complemented with these deletion variants, the DNase activity was generated. Among these variants, the highest DNase activity generated was when the C-terminal 10 residue-deleted brDNase(��251-260) was incubated with the C-terminal 10 residue-peptide (Peptide C10) in a molar ratio of 400 equivalents at room temperature for 8 h. The non-covalent binding of Peptide C10 with brDNase(��251-260) exhibited a dissociation constant of 48 �嵱. Circular dichroism spectra showed that the deletion mutants were partially folded with mainly helical structures, and that admixture with corresponding peptides facilitated their folding into the native-like ��-sheet-rich structure. Thermal denaturation profiles also revealed that the Tm for brDNase(��251-260) was 55oC, while the Tm increased to 63 oC upon incubation with Peptide C10, very close to the Tm of bpDNase (65 oC). The calcium ion was essential for the two-stage folding-activation process.
42
SHEN, TE-JEHN y 沈德政. "Site-directed Mutagenesis of Bovine Pancreatic Phospholipase A2 : Correlation of Enzyme Activity and Neurotoxicity with Mutation at Positions 22 , 113 , 115 and 118". Thesis, 1997. http://ndltd.ncl.edu.tw/handle/55357886917073985963.
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碩士國立臺灣大學
生化科學研究所
85
Site-directed mutagenesis was used to generate mutants of bovine pancreatic phospholipase A2 for studying the structure and functions of the protein. The pTO-A2M plasmid , which bovine pancreatic PLA2 gene has been inserted into ,was used as template and designed oligonucleotide fragments as primers , in polymerase chain reaction to obtain cDNA for PLA2 with desired mutation sites. The DNA was inserted into the expression vector, pTO-A2M, and the plasmid was transfected into the E.coli DE3 strain . The expressed PLA2 mutant proteins were extracted , correctly refolded and then purified by ion exchange chromatography and reversed phase HPLC. In this way , five mutant proteins were obtained. They are F22W, K113Y ,H115Y, L118Y and H115Y + L118Y. The enzyme activity of each mutant protein was assayed by the pH-stat method using egg lecithin as substrate. The mutant protein F22W completely lacks PLA2 enzyme activity, and the other mutant proteins show little change in the enzyme activity . All five mutants are unable to compete in both equilibrium binding assay and cross-linking experiment with 125I-Taipoxin for binding to synaptic membrane proteins.
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Kim, Ok-Hee. "Mass spectrometric studies of peptides and proteins : probing structural elements and structural fluctuations in melittin and bovine pancreatic trypsin inhibitor (BPTI) using amide H/D exchange and HPLC-electrospray ionization mass spectrometry". Thesis, 1996. http://hdl.handle.net/1957/34509.
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