Дисертації з теми "Peptide resonance"
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1
Costa, Philip R. (Philip Remi). "Spins, peptides, and Alzheimer's disease : solid-state nuclear magnetic resonance investigations of amyloid peptide conformation." Thesis, Massachusetts Institute of Technology, 1996. http://hdl.handle.net/1721.1/10714.
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Mozsolits, Henriette 1971. "Surface plasmon resonance spectroscopy for the study of peptide-membrane interactions." Monash University, Dept. of Biochemistry and Molecular Biology, 2001. http://arrow.monash.edu.au/hdl/1959.1/8123.
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Thirumoorthy, Ramanan. "NMR studies of structure and dynamics of novel peptide-based melanocortin receptor antagonists." [Gainesville, Fla.] : University of Florida, 2002. http://purl.fcla.edu/fcla/etd/UFE1001186.
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Drew, Daniel L. Jr. "Investigating the Structure and Dynamic Properties of Bacteriophage S21 Pinholin Using Solid-State Nuclear Magnetic Resonance and Electron Paramagnetic Resonance Spectroscopy." Miami University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=miami1610187893016095.
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Smith, K. I. "The two-dimensional nuclear magnetic resonance spectroscopy analysis of peptides in solution." Thesis, University of Manchester, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377720.
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Brown, Peter N. "Biophysical and structural characterisation of protein-peptide interactions." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/3982.
Повний текст джерелаАнотація:
Proliferating cell nuclear antigen (PCNA) is an essential protein in the cell. It is involved in transcription and many types of DNA repair and replication. Homologues of this protein are found in all orders of life. The high level of conservation and essential nature of PCNA infers that it may be a potential drug target for anti-caner drugs in humans and also a potential anti-parasitic target. X-ray structures of PCNA from Homo sapiens (Hs), Schizosaccharomyces pombe (Sp) and Leishmania major (Lm) are now available and can be used as a template for structure based drug design. In this work PCNA from these three species have been prepared in milligram quantities for biochemical and biophysical studies. The previously unknown structure of LmPCNA has been solved in an uncomplexed form and also complexed with a dodecapeptide to a resolution of 3.0Å. A comparison of PCNA structures and their peptide complexes for the three species identifies structural differences which may be relevant in analysing thermodynamic contributions of binding. All eukaryotic PCNA molecules exist as ring shaped trimers which form around DNA. In this work the oligomeric state of LmPCNA has been determined to be hexameric both in solution and in the crystal. It has also been hypothesised that HsPCNA is hexameric however these would seem to form hexamers in which the trimeric rings associate “back-to-back” while LmPCNA trimers would seem to associate “face-to-face”. The binding affinities for these three PCNAs have been determined with a selection of peptides derived from the Hs p21 protein. This work has shown, using a selection of different techniques including Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC) and Dynamic Scanning Fluorimetry (DSF); that HsPCNA and SpPCNA have similar affinities for a 12mer peptide (Kd of ~1μM) however LmPCNA shows significantly weaker interactions (Kd of ~10μM). This is most likely due to divergence in the sequence and structure of LmPCNA. A systematic investigation by SPR on the effect of peptide linker length on binding has been carried out using a series of synthesised peptides with different lengths of chemical spacer. The series of streptavidin immobilised peptides show that longer spacers are required for the recovery of the PCNA peptide binding affinity. The results presented in this work indicate that a linker length of at least 20Å is required for measurable protein binding activity. This interaction is improved with longer peptide spacers.
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DUPONT-QUENET, BEATRICE. "Etude par resonance magnetique nucleaire du peptide a13g. Recherche de l'interaction avec l'alprenolol." Paris 6, 1994. http://www.theses.fr/1994PA066358.
Повний текст джерелаАнотація:
Le peptide a13g est la representation synthetique d'une region determinante d'un anticorps anti-alprenolol: 37a4. Dans le but de confirmer l'interaction entre ces deux molecules, nous avons observe, par resonance magnetique nucleaire, le comportement du peptide seul et en presence d'alprenolol, en solution aqueuse. L'attribution complete du spectre proton et partielle en carbone a ete realisee. Nous avons constate, qu'a ph acide, celui-ci s'hydrolysait au bout de 5 jours au niveau de la liaison asp-pro. L'etude conformationnelle n'a pas permis de mettre en evidence une structure secondaire reguliere du peptide. Seule, la configuration trans des deux liaisons x-pro a pu etre demontree. Le peptide presente les memes caracteristiques lorsqu'il est en presence d'alprenolol. L'etude de l'interaction, a travers des mesures d'effets overhauser nucleaires intermoleculaires et de temps de relaxation des protons de l'alprenolol, a montre que l'alprenolol ne reconnaissait pas le peptide a13g. Ce dernier serait une representation trop restreinte de l'anticorps anti-alprenolol
8
Neidigh, Jonathan Wesley. "Chemical shift tools in peptide folding and miniature protein design /." Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/8701.
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Francart, Céline. "Etude par resonance magnetique nucleaire de polypeptides contenant plusieurs prolines." Lille 2, 1997. http://www.theses.fr/1997LIL2P269.
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PRECHEUR-AGULHON, BENEDICTE. "Etudes conformationnelles par resonance magnetique nucleaire et par dichroisme circulaire de peptide d'interet biologique." Paris 11, 1993. http://www.theses.fr/1993PA112345.
Повний текст джерелаАнотація:
Dans une premiere etude j'ai entrepris l'analyse conformationnelle de peptides issus du domaine d'interaction des adenylates cyclases de bordetella pertussis et de bacillus anthracis, avec la calmoduline. Les resultats obtenus suggerent que chacun de ces peptides a tendance a se replier en helice- amphiphile basique, proposee comme motif structural pour l'interaction avec la calmoduline. Afin de confirmer ce modele d'interaction, j'ai entrepris l'etude conformationnelle d'un peptide modele construit pour former une helice- amphiphile basique. L'analyse combinee des experiences rmn homo- et heteronucleaires ont de plus permis l'identification du site d'interaction sur la calmoduline. Dans une seconde etude nous avons caracteriser la conformation d'un peptide antigenique de echinococcus granulosus. Les donnees spectroscopiques obtenues sur le peptide suggerent fortement la presence de trois tours d'helice-. Nous avons tente de stabiliser l'organisation helicoidale du peptide afin d'augmenter l'activite biologique. Toutefois, les etudes conjointes par rmn et par dc montrent que la stabilisation helicoidale est fortement reduite
11
Cao, Yihong. "Sugar and Peptide mimics for SPR Characterization of autoantibodies in monoclonal gammopathy." Phd thesis, Université de Cergy Pontoise, 2013. http://tel.archives-ouvertes.fr/tel-00877262.
Повний текст джерелаАнотація:
IgM monoclonal gammopathy is a common age-related demyelinating sensory and motor polyneuropathy. It has been shown to be associated with antibodies against myelin-associated glycoproteins (MAG/SGPG). The HNK-1 carbohydrate epitope is a terminal 3-sulfo-glucuronyl residue attached to lactosamine structures and it is shared both in MAG and SGPG (SO4-3-GlcA(β1-3)Gal-(β1-4)GlcNAc(β1-3)Gal-(β1-4)Glcβ(1-1′)Cer). It is mostly expressed in the nervous system and plays an important role in preferential motor reinnervation. Nevertheless, the HNK-1 epitope is difficult to be isolated and synthesized and diagnostic assays used in the clinics are not always reproducible and reliable. Therefore in our study, our goal is to identify a simple synthetic diagnostic tool (peptide or monosaccharide), mimetic of the HNK-1 epitope, able to recognize antibodies in neurogammopathies sera by Surface Plasmon Resonance to be used in earlier stage patients and possibly to monitor disease activity. For this reason, we firstly tried to synthesize this trisaccharide and then we achieved the synthesis of its terminal monosaccharides with different function groups (octyl glucopyranoside, octyl glucuronic acid, octyl 3-O-sulfo-glucuronic acid and 8-amino octyl 3-O-sulfo-glucuronic acid). Then 10 linear and cyclic peptides conformationally and/or structurally mimicking HNK-1 were also synthesized (LSETTI, LSETTl, cyclo(-TTILSE-), cyclo(-TTlLSE-), cyclo(-TKTlLSE-), cyclo(-TETKlLSE-), TYTKlLSE, TY(SO3)TKlLSE, cyclo(-TYTKlLSE-) and cyclo(-TY(SO3)TKlLSE-)). The SPR kinetic binding affinities of all these sugar and peptide mimics were studied with commercial anti HNK-1 antibody using Biacore. Moreover, mimics with highest binding affinities were chosen for antigen-antibody interaction study in IgM gammopathy patients' serum.
12
Bedrossian, Diane. "The effects of ghrelin on the amygdala response to visual food and non-food stimuli : an fMRI study in humans." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111507.
Повний текст джерелаАнотація:
A complex physiological system, composed of central and peripheral signals, balances energy intake and expenditure. Among these signals, the enteric and orexigenic hormone ghrelin is a regulator of energy balance with several uncharacterized functions. Although much research has accumulated regarding ghrelin's effects on metabolic parameters, little is known about its other behavioural and cognitive effects. Consequently, this study, using functional magnetic resonance imaging, showed that ghrelin administered intravenously to healthy volunteers increased the neural response to food pictures, as well as faces of fear and disgust, in brain areas regulating the hedonic and incentive evaluation of visual stimuli, such as the amygdala. Moreover, ghrelin exhibited memory enhancing effects for both food pictures and faces of fear and disgust. These findings suggest that ghrelin's activation of the amygdala may serve as a magnitude signal for value judgments of visually-presented food and non-food stimuli, thus engaging critical feeding, emotional and cognitive responses.
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CODY, WAYNE LIVINGSTON. "SYNTHESIS, BIOLOGICAL ACTIVITY AND CONFORMATIONAL ANALYSIS OF FRAGMENT ANALOGUES OF ALPHA-MELANOTROPIN (PEPTIDE, STRUCTURE-FUNCTION, PHENYLGLYCINE, NMR, TETRAHYDROISOQUINOLINE-3-CARBOXYLATE)." Diss., The University of Arizona, 1985. http://hdl.handle.net/10150/188044.
Повний текст джерелаАнотація:
α-MSH (α-melanotropin) is a naturally occurring linear tridecapeptide (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) that is primarily known for its ability to stimulate integumental melanocytes and more recently has been implicated in a variety of physiological and neurological processes. It has been shown that substitution of D-phenylalanine in the seven position of this hormone led to an analogue with increased potency and prolonged biological activity. Furthermore, cyclization between the four and ten positions via a cystine bridge led to analogues with enhanced potency. In this regard, a series of conformationally restricted linear and cyclic fragment analogues of α-MSH have been prepared and carefully analyzed by both biological and biophysical methods. Conformational restriction was incorporated in α-MSH fragment analogues, by: (1) substitution of sterically restricted amino acids into the native sequence; or (2) cyclization of the peptide via a disulfide bridge. Due to the biological differences observed for these synthetic α-MSH fragment analogues, a complete conformational analysis by both proton and carbon-13 NMR was performed. The conformational preferences of the backbone were examined by analyzing: (1) the alpha proton chemical shifts; (2) the amide proton chemical shifts; (3) the amide proton coupling constants; and (4) the amide proton temperature dependencies. The data suggests that the peptide backbone in both linear and cyclic analogues possesses a great amount of conformational flexibility with no hydrogen-bonded stabilization. The three-dimensional orientations of individual amino acid side chains have been examined by analyzing: (1) the chemical shifts of the side chain protons; (2) the alpha-beta coupling constants (corresponding rotamer populations); and (3) the carbon-13 spin lattice relaxation times (T₁). A careful examination a the chemical shifts of the side chains of individual amino acids in linear α-MSH fragments reveals that incorporation of an aromatic D-amino acid in the seven position results in an interaction of the side chains of the six, seven and eight positions. In addition, the low carbon-13 spin-lattice relaxation times for the β-carbons of the 5-9 sequence for both Ac-[Nle⁴]-α-MSH₄₋₁₁-NH₂ and Ac-[Nle⁴, D-Phe⁷]-α-MSH₄₋₁₁-NH₂, provides further evidence for an interaction of these side chains. Similar shielding patterns have been observed for the cyclic α-MSH fragment analogues depending upon whether L- or D-phenylalanine is incorporated in the seven position. Considering the differences in biological potency and the similarities in the NMR parameters between the linear and cyclic homologs, it can be concluded that the conformational properties that determine biological potency are too subtle to be measured by present NMR methodology. Furthermore, the similarity of the NMR shielding patterns suggests that a 23-membered ring is too large to impart significant conformational constraints on the peptide backbone or amino acid side chains.
14
Palmblad, Magnus. "Identification and Characterization of Peptides and Proteins using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry." Doctoral thesis, Uppsala universitet, Institutionen för materialvetenskap, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-1999.
Повний текст джерелаАнотація:
Mass spectrometry has in recent years been established as the standard method for protein identification and characterization in proteomics with excellent intrinsic sensitivity and specificity. Fourier transform ion cyclotron resonance is the mass spectrometric technique that provides the highest resolving power and mass accuracy, increasing the amount of information that can be obtained from complex samples. This thesis concerns how useful information on proteins of interest can be extracted from mass spectrometric data on different levels of protein structure and how to obtain this data experimentally. It was shown that it is possible to analyze complex mixtures of protein tryptic digests by direct infusion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and identify abundant proteins by peptide mass fingerprinting. Coupling on-line methods such as liquid chromatography and capillary electrophoresis increased the number of proteins that could be identified in human body fluids. Protein identification was also improved by novel statistical methods utilizing prediction of chromatographic behavior and the non-randomness of enzymatic digestion. To identify proteins by short sequence tags, electron capture dissociation was implemented, improved and finally coupled on-line to liquid chromatography for the first time. The combined techniques can be used to sequence large proteins de novo or to localize and characterize any labile post-translational modification. New computer algorithms for the automated analysis of isotope exchange mass spectra were developed to facilitate the study of protein structural dynamics. The non-covalent interaction between HIV-inhibitory peptides and the oligomerization of amyloid β-peptides were investigated, reporting several new findings with possible relevance for development of anti-HIV drug therapies and understanding of fundamental mechanisms in Alzheimer’s disease.
15
Lai, Pok-man, and 黎博文. "Structure determination of N-terminal peptide of nucleoprotein (NP20) of influenza virus H5N1 by nuclear magnetic resonance spectroscopy." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hub.hku.hk/bib/B50899958.
Повний текст джерелаАнотація:
Influenza virus has long been a major threat to public health worldwide. The virus can be highly deadly because of antigenic shift. Since the H5N1 outbreak in Hong Kong in 1997, avian flu is regarded as the next pandemic threat. For combating the disease, it is essential to investigate more on the influenza virus, in particular H5N1. Nucleoprotein (NP) is a major component of the ribonucleoprotein complex (RNP) in the influenza virus. NP exhibits both structural and functional roles for influenza virus assembly and propagation and is involved in mediating the transcription-replication process. The NP of the virus binds the RNA genome and acts as a key adapter between the virus and the host cell. It therefore plays important roles and represents an attractive drug target. Recently, the X-ray structure of H5N1 NP was solved to a resolution of 3.3 Å , which provides valuable clues on how NP carries out its functions. However, the N-terminal 1-20 residues were not resolved in the H5N1 NP crystal structure. This N-terminal region is thought to contain a nuclear localization signal (NLS), a cellular splicing factor BAT1/UAP56 binding site, and a nuclear export signal. It has been suggested that the N-terminal NLS binds to importin (a cytosolic protein) for the nuclear import of NP.
In the present study, the solution structure of H5N1 NP N-terminal peptide (NP20) in membrane mimetic solvent condition was determined using Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopies. The CD results show that NP20 adopted an α-helical conformation. The NMR data indicate that NP20 formed a single α-helix spanning from residues Gly5 to Gly16. Surface electrostatic potentials further showed that the NP20 peptide is amphipathic in nature, which may be important for its binding with importin. NMR titration experiments have been carried out between NP20 and importin. Addition of importin into the solution of NP20 peptide caused significant broadening of the NMR signals of NP20 and progressive changes of the chemical shifts of NOE cross-peaks at increasing importin concentration confirm that NP20 could bind with importin. Therefore, the present study supports that NP20 region is the binding site of importin mediating the import of NP into the host cell nucleus.
In conclusion, the knowledge gained from this study provides a better understanding on the structure of NP20 and its interaction with the host importin protein, and may serve as a template for the development of novel antiviral drug targeting NP with improved therapeutic index.published_or_final_version
Chemistry
Master
Master of Philosophy
16
Raghunathan, Vinodhkumar. "Elucidation of molecular recognition mechanisms of a peptide involved in biomineralization using solid state nuclear magnetic resonance spectroscopy /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/8644.
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Liu, Xiaohua. "Anion-Peptide Adduct Formation and Decomposition As Studied by Fourier Transform Ion Cyclotron Resonance (FT-ICR) Mass Spectrometry." ScholarWorks@UNO, 2013. http://scholarworks.uno.edu/td/1748.
Повний текст джерелаАнотація:
A new “best match” match model has been developed to account for adduct formation on multiply charged peptides observed in negative ion electrospray mass spectrometry. To obtain a stable adduct, the model necessitates an approximate matching of apparent gas-phase basicity (GBapp) of a given proton bearing site on the peptide with the gas-phase basicity (GB) of the anion attaching at that site. Evidence supporting the model is derived from the fact that singly charged adducts were only observed for lower GB anions: HSO4-, I-, CF3COO-. Ions that have medium GBs (NO3-, Br-, H2PO4-) only form adducts having -2 charge states, whereas Cl- (higher GB) can form adducts having -3 charge states.
Hydrogen bonds are the main interactions pertinent to the “Best Match” model, however, ion-ion interactions formed between peptides ([Glu]Fibrinopeptide B, Angiotensin I or [Asn1,Val5]-Angiotensin II) and low GB anions (ClO4- or HSO4-) have been established by CID-MS/MS. Evidence for ion-ion interactions comes especially from product ions formed during the first dissociation step, where, in addition to the expected loss of the anion or neutral acid, other product ions that require covalent bond cleavage (i.e., H2O or NH3 loss) are also observed.
In this study, the “Best Match” model is further supported by the decomposition behavior of adducts formed when Na+/H+ exchange has occurred on peptides. Na+/H+ exchanges were found to occur preferentially at higher acidity sites. Without any Na+/H+ exchange, F- and CH3COO- can hardly form observable adducts with [Glu]Fibrinopeptide B. However, after multiple Na+/H+ exchanges, F- and CH3COO- do form stable adducts. This phenomenon can be rationalized by considering that Na+ cations serve to “block” the highly acidic sites, thereby forcing them to remain overall neutral. This leaves the less acidic protons available to match with higher GB anions.
According to the "best match" model, high GB anions will match with high GBapp sites on the peptide, whereas low GB anions will match with low GBapp peptide sites. High charge states readily augment GBapp of the peptide (through-space effect). Na+/H+ exchanges substantially decrease GBapp by neutralizing charged sites, while slightly increasing intrinsic GBs by the inductive effect.
18
Lambert, Matthew Alexander. "Using B-type natriuretic peptide and whole body contrast enhanced magnetic resonance imaging to detect asymptomatic cardiovascular disease and improve prediction of risk of cardiovascular disease : the TASCFORCE Study." Thesis, University of Dundee, 2016. https://discovery.dundee.ac.uk/en/studentTheses/7bc498a0-4c96-4eab-9a2b-99810c032824.
Повний текст джерелаАнотація:
Cardiovascular disease remains a leading a cause of mortality and morbidity. Primary prevention is known to reduce the incidence of cardiovascular disease. The use of medication is currently targeted at those at increased predicted risk of cardiovascular disease using risk prediction tools developed from large epidemiological studies. However these have poor external validity particularly for those at low or intermediate risk: a significant number of cardiovascular events still occurs in these groups. We hypothesised that screening for asymptomatic pre-clinical cardiovascular disease using B-type natriuretic peptide (BNP) and whole body contrast enhanced magnetic resonance imaging (MRI) could identify those at low/intermediate risk or disease whowill develop clinical disease and thus facilitate improved targeting of primary prevention at those most likely to benefit. The Tayside Screening for Cardiac Events (TASCFORCE) study is a prospective normal volunteer cohort study. Men and women aged 40 years or older free from cardiovascular disease and with a predicted 10-year coronary heart disease risk less than 20% were recruited. All had comprehensive baseline cardiovascular risk information and a BNP level measured. If the BNP level was greater than the median for their gender participants were invited to attend for a whole body contrast enhancedMRI scan comprising cardiac imaging and whole body angiography. The images were analysed to measure left ventricular mass (LVM), left ventricular volumes and left ventricular function. These were indexed for body size using height, height1.7, height2.7 and body surface area. Angiogram images were analysed for the presence and degree of intraluminal stenosis. All participants are being followed up using anonymised electronic data linkage for incident cardiovascular disease and death. 4423 participants (39.3% male) were recruited between November 2007 and February 2013. Median age was 51.2 years. The median 10-year coronary heart disease (CHD) 23 risk was 2% and 13.6% had a CHD risk of 10-19.9% (intermediate risk). The medianBNP results for men and women were 7.5 and 15.3 pg/ml respectively. Age, female sex and high density lipoprotein were independently associated with BNP level. Heart rate, total cholesterol and ex-smoking status were independently inversely associated with BNP level. 1528 (74.8% of those invited) underwent an MRI scan. Mean left ventricular mass was 129.2g and 87.0g for men and women respectively. LVM and left ventricular mass index (LVMI) were significantly higher in men than women. The vast majority (94.6%) of arterial segments analysed were normal and 50.6% of individuals had no evidence of luminal stenosis. From follow up data obtained 2 years after the end of recruitment 18,364 person years at risk were analysed. 17 cardiovascularevents and no deaths occurred in those not invited for an MRI scan based on their BNP result and 16 events and 1 death occurred in those invited for an MRI scan. There was no significant difference in event rates between those with above and below median BNP levels, between those with higher or lower LVM or LVMI or between those with and without the presence of stenosis on angiography. As expected we have not demonstrated the ability of LVM, LVMI or stenosis burden determined using magnetic resonance imaging to predict cardiovascular disease in a population at low or intermediate risk of CHD. We have also not demonstrated the ability of BNP to identify those at low orintermediate risk of CHD who will develop clinical CV disease. However it is the pre-planned longer-term follow up where difference might be expected. The low number of events at this early stage in follow up mean that it is difficult to draw firm conclusions. As follow up continues and further events accumulate we hope to determine if these measures will be shown to predict cardiovascular events in future analyses. We have characterised the normal values and distribution of a range of left ventricular structural and functional parameters derived using a steady state free precision sequence MRI in a population at low or intermediate risk of CHD which will provide a useful reference for normal values that are different to other imaging modalities including chocardiography and other protocols of MRI scanning.
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Suñol, Moreno David. "Structural studies of recombinant TGIF1 and FBP28 WW domains using NMR and peptide ligation strategies." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/400299.
Повний текст джерелаАнотація:
The present thesis is divided in three different but related projects. In the first project, the interaction between TGIF1 and SMAD proteins is investigated. TGIF1 is a transcriptional suppressor that prevents the gene transcription due to its interaction with DNA and protein suppressor complexes. TGIF1 has a role in different signalling pathways such as retinoid, Wnt or TGF-β. Focusing on TGF-β, this is one of the main signalling pathways that regulates a plethora of functions in metazoans, including cell differentiation, proliferation and tissue homeostasis, among many others. SMAD proteins are the centrals mediators of the pathways, carrying the signal from the TGF-β receptors at the plasma membrane to the DNA. The aim of this project was to describe, from a structurally point of view, the interaction between TGIF1 and SMAD proteins, already detected biochemically. Using NMR spectroscopy and EMSA assays we could observe the interaction between TGIF1 and the MH1 domain of SMAD2 and SMAD4. Furthermore, the addition of SMAD2/4-MH1 disrupts the TGIF1-DNA interaction. On the other hand, the binding between SMAD2-MH2 and TGIF1 (256-347) was not detected in our experimental conditions.
Moreover, we found that p38α and CK1 kinases phosphorylate serines 286, 291 and 294 of TGIF1 (256-347) in vitro. While these serines are located in a region where TGIF1 interacts with many proteins - including SMAD2, HDAC, or Axin-2 - , the phosphorylation could indicate a regulation mechanism. However, the phosphorylation does not change the overall structure of the TGIF1 fragment.
Finally, we have detected an interaction between TGIF1 fragments 150-248 and 256-347, suggesting the presence of open and closed conformations of full-length TGIF1.
The second project is related to the ligation reaction between two peptides. Our study demonstrates that the addition of HOBt to the cysteine-free direct aminolysis ligation reaction between one thioester peptide and one N-terminal free peptide increase the conversion but not the rate of the ligation. This effect is especially increased when sterically hindered amino acid (such as valine or leucine) are present in the ligation junction. The reaction is also compatible with phosphorylated peptides but intramolecular cyclisation side-reactions appear when the peptide thioester is not protected at the N-terminus.
Lastly, six selected mutants structures of FBP28-WW2 were determined de novo by NMR spectroscopy. The six mutant structures maintain the main characteristics of WW domain structure, even for the mutations introducing deletions at the N and C termini. The structures were the experimental confirmation of the effect of this set of mutations in the structure. The existence of the WW fold in all these mutations was key to provide the grounds for the simulated folding curves generated using the UNRES force field.TGF-β és una de les principals vies de senyalització que tenen les cèl·lules animals per regular gran part dels processos cel·lulars, tals com la diferenciació i proliferació cel·lular o la regeneració, entre d’altres. Les proteïnes SMAD són les principals mediadores d’aquesta via, portant la senyal des dels receptors de TGF-β situats a la membrana cel·lular fins al DNA a l’interior del nucli. En la present tesi s’ha dut a terme un estudi sobre la interacció entre el factor de supressió TGIF1 i les proteïnes SMAD2 i SMAD4. Mitjançant ressonància magnètica nuclear s’ha demostrat la interacció entre TGIF1 i el domini MH1 de SMAD2 i SMAD4. A més, aquesta unió interromp el contacte entre TGIF1 i el seu DNA canònic tal com demostren els EMSA efectuats. Altrament, s’ha determinat per primera vegada que TGIF1 és un substrat de les quinases p38α i CK1, que fosforilen les serines 286, 291 i 294, localitzades en una regió d’interacció amb diverses proteïnes, com per exemple SMAD2, HDAC o Axin-2. D’altra banda, en aquest treball s’ha estudiat la lligació nativa de dos pèptids mitjançant una reacció d’aminòlisis directa sense la presència de cisteïnes en el lloc d’unió. Específicament, s’ha demostrat que l’addició de HOBt a la mescla de reacció augmenta la conversió però no la velocitat de la lligació entre els dos pèptids. Aquest increment en la conversió és especialment rellevant quan aminoàcids estericament impedits, com valina o leucina, es troben en el lloc d’unió. Finalment, s’han determinat les estructures de 6 mutants del domini WW2 de FBP28. Tots els mutants conserven el plegament característic dels dominis WW tot i les delecions que presenten tant a C com a N-terminal. Les estructures han servit per confirmar els resultats de simulacions moleculars efectuades mitjançant el camp de força UNRES.
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Thomas, Celestine J. "Endotoxin Peptide/Protein Interactions: Thermodynamic And Kinetic Analysis." Thesis, Indian Institute of Science, 2000. http://hdl.handle.net/2005/213.
Повний текст джерелаАнотація:
Endotoxin or Lipopolysaccharide (LPS) is the invariant structural component of gram negative bacterial outer membranes and is the chief causative factor of Sepsis or endotoxic shock. Sepsis is a syndrome that has very high mortality rates even in this age of excellent therapeutics and critical patient care. The treatment for sepsis till date remains nonspecific and supportive due to lack of effective anti-endotoxic drugs. Sepsis is initiated when the circulating bacteria shed LPS from their cell envelopes. Shed LPS aggregates are recognized by LPS binding proteins and receptors, which activate the host's immune system. Uncontrolled and excessive stimulation of the host's immune system precipitates endotoxic shock which in advanced cases involving multiple system organ failure inevitably lead to patient's death.
Many strategies have been tested out to combat this deadly affliction. One of the attractive clinical modalities in sepsis treatment is the use of peptides as LPS sequestering anti-endotoxic drugs. A classical peptide antibiotic of this class is Polymyxin B (PMB) a cyclic cationic acylated molecule, that recognizes LPS with a very high affinity.
This thesis describes kinetics and thermodynamics of PMB-LPS interactions and applies these parameters over a framework of different models so as to gain insights into the structure-function relationships that govern the interactions of this peptide with endotoxin(s). Classical biophysical techniques like fluorescence, circular dichroism spectroscopy, stopped flow kinetics, titration calorirnetry (ITC) and the relatively new technique of Surface Plasmon Resonance (SPR) have been employed to dissect out the mechanism of the range of non-covalent forces that are involved in peptide-endotoxin recognition. Certain proteins that exhibit LPS binding activity have also been studied to gains insight about their mode of action. Implications of these studies for designing peptides that have better anti-endotoxic properties are also highlighted.
The first chapter introduces and highlights the clinical features of sepsis. It also attempts to shed light on the LPS mediated signal transduction pathway that leads to endotoxic shock. This chapter also briefly explains the roles of many LPS receptors that are present in the human system and their specific roles in the signal transduction pathways.
The second part of this chapter deals with the role of cationic peptides as anti-endotoxic drugs. Certain key functional aspects of these peptides, which impart in them, the desirable property of LPS recognition have also been discussed
The second chapter describes the kinetic studies undertaken to unravel the exact mechanism of LPS-PMB interaction. The studies reveal that PMB recognizes LPS in a biphasic manner, with the second, unimolecular isomerization step of the reaction being the rate-limiting step. The initial reaction is shown to be influenced by the presence of salt in the reaction medium. The dissociation phase of this interaction also shows a biphasic pattern. These data allow us to speculate upon the exact mechanism by which PMB is able to recognize LPS. The studies also shed light on some structural aspects that govern and confer such high LPS binding activity to PMB. Based on these a model has been proposed to explain this recognition (C.J. Thomas et al, 1998).
The second chapter discuses the mode of action of various PMB analogs. These analogs have been chosen in terms of their mode of action as well as their structural similarly to PMB. The affinities of these analogs to LPS and lipid A were quantified using the Surface plasmon resonance (SPR) method. SPR, a technique that relies on the quantification of change in mass during a binary binding process occurring between an immobilized entity and a flowing ligand, is a rapid and sensitive method to measure biologically relevant interactions.
SPR studies provide us with the binding constants and thermodynamic parameters that allow evaluation of the affinities of these peptides towards LPS (C.J.Thomas and A.Surolia, 1999).
The third chapter discusses a hitherto unknown mode by which PMB acts on a LPS lamellae. The results of this study wherein the binding affinities of PMB and its analogs were performed on monolayers and tethered liposomes, show that PMB is able to remove specifically LPS or lipid A from monolayers or bilayer assemblies such as tethered liposomes. The exact mode of action of PMB is deciphered in the light of these new studies, which allow us to posit on the observed efficacy of PMB in neutralizing the endotoxin as compared to peptides with nearly similar affinities for LPS (C.J Thomas et al 1999).
In the fourth chapter a series of 23 residue peptides, based on the sequence corresponding to the anti-sense strand of magainin gene have been synthesized. Magainin an amphiphilic helical peptide obtained from frog skins plays a vital role in the innate immune defense mechanisms of these organisms. It also exhibits LPS binding activity that makes it an attractive target as an anti-endotoxic drug. Biochemical and biophysical characterization of these peptides reveal that they have the tendency to perturb both the inner and the outer membranes of E.coli. The peptides are amphiphilic and have helical structure in a membrane bound environment.
Three of the peptides tested have high affinities for lipid A that approach the values shown by PMB. The kinetic parameters obtained by stopped flow and SPR studies in conjunction with the therrnodynamic parameters obtained using ITC studies allow us to highlight the key structural features that need to be exhibited by peptides that are designed to be LPS recognizers. The studies also project the fact that ionic forces play an important role in the initial recognition of LPS by these peptides. Fortification of the might of these ionic charges increases affinity for LPS where as the hydrophobic residues that interact at the next phase of binding are more amenable to disruptions in contiguity. These factors are discussed using the helical wheel diagram that shows the clear amphiphilicity displayed by these peptides. (C.J Thomas et al Manuscript under preparation, 2000)
Chapter six discusses the mode of action of certain LPS binding proteins. Limulus anti endotoxic factor (LALF) plays a vital role in the innate immune based defense systems of the horseshoe crab. Galectin-3 is a metal ion independent, galactosc binding Icctin of human origin with unknown functions. Both these phylogcntically-unrclatcd proteins exhibit LPS/lipid A recognizing properties. ITC and SPR studies have been used to determine the binding constants displayed by these proteins for lipid A. LALF bind to lipid A with very high affinity than compared to Galectin-3 and is also able to take away selectively lipid A from both monolayers and tethered liposomes. Galectin-3 does not show this property of LALF, which might account for its lowered affinities. Also structurally LALF has amphiphilic nature that confers high lipid A binding activity, which is clearly lacking in Galectin-3. These studies in conjunction with the knowledge gained from the study of LPS-PMB interaction stress on the importance of amphiphilicity in LPS recognition. (C.J Thomas et al Manuscript under preparation, 2000).
The final chapter is a general discussion that attempts to collate all these kinetic and thermodynamic observations in the pursuit of designing small easily manipulatable peptides that exhibit high LPS binding activity. These studies are aimed to act as rough guidelines to the design of LPS sequestering peptides that might have better therapeutic and pharmacokinetic properties.
The appendix to the main body of work presented in thesis are two pieces of work pertaining to the elucidation the kinetics and mechanism of sugar lectin interactions, when sugars are presented as glycolipids in monolayers or bilaycrs liposomes. Mode of the presentation of sugars at cell-surfaces in the form of glycolipids as ligands influence their recognition by macromolecular receptors like lectins. Appendix 1 is a study of the mode of action of Ulex europeus I lectin binding to H-fucolipid containing tethered liposomes, by SPR. Fucosylated sugars are often used as key markers in histochemical analysis of malignant cancerous tissues. Ulex lectin plays a vital role as a marker for identification of these tissues. The kinetics and thermodynamic parameters that are obtained in this study throw some light on the mode of recognition of glycolipid receptor by Ulex europeus I lectin (C.J Thomas and A. Surolia 2000).
Appendix 2 is a study, that attempts to quantify the initial kinetic parameters that correlate the recognition of glycolipid receptors with their inclination at the membrane surface and the influence of charge on them by soyabean agglutinin (SBA), Abrus agglutinin I and II. Studies on the soyabean agglutinin-globoside interaction highlights the divalent cation mediated reorientation of these receptors on their accessibility and recognition to the agglutinin. The divalent cations are speculated to orient the oligosaccharide head groups in a spatial geometry that allows a heightened kinetics of their interaction by SBA. These studies reveal that the reorganization of the binding pocket of a lectin can also have a profound influence on ihc rates of recognition of a glycospingolipid ligand by a lectin as exemplified by Abrus agglutinin II- GM1 interactions (C.J Thomas ct al, Manuscript under preparation).
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Betts, Corinne A. "Exon skipping peptide-pmos for correction of dystrophin in mouse models of duchenne muscular dystrophy." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:545d586a-ad7b-4089-8537-b2677957b874.
Повний текст джерелаАнотація:
Duchenne muscular dystrophy (DMD) is a fatal, muscle-wasting disorder due to mutations/deletions in the dystrophin gene. Whilst improvements in palliative care have increased the life expectancy of patients, cardiomyopathy and respiratory complications are still the leading causes of death. A potential therapy for the treatment of DMD is antisense oligonucleotides (AOs), which modulate dystrophin pre-mRNA splicing to restore the dystrophin reading frame and generate a truncated functional protein. Conjugation of AOs to cell penetrating peptides (CPP), such as Pip5e-, significantly improves delivery to skeletal muscles and to the heart, which is imperative given the impact of cardiomyopathy to mortality. However, it should be noted that the contribution of skeletal muscles, such as the core respiratory muscle, the diaphragm, in dystrophic cardiopulmonary function is poorly understood. The specific aims of the work in this thesis were to (i) understand the effect of the diaphragm on cardiac function using magnetic resonance imaging (MRI), (ii) screen a number of derivatives of Pip5e (Pip6) in an effort to discover further promising peptides and define the properties integral to heart penetrating capacity, and (iii) assess whether Pip6-PMOs restore cardiac function (MRI) following a repeat, low dose regimen. In short, the specific restoration of dystrophin in the diaphragm of the dystrophic mouse model, the mdx mouse, did not improve cardiac function, highlighting the importance of a body-wide therapy. The screening of multiple Pip5e-PMO derivatives revealed 3 promising peptides with improved cardiac splicing capacity; however, serial deletions of amino acids from the central core resulted in the diminution of dystrophin restoration, possibly due to a reduction in hydrophobicity. Finally, the Pip6-PMO treatment regimen substantially restored dystrophin protein (28% in heart) and stabilised cardiac function, even with an increased work load. In conclusion, this study illustrates the importance of a body-wide treatment, such as the CPP strategy (Pip-PMO). These Pip-PMO conjugates demonstrate high dystrophin restoration in a number of muscles, including cardiac muscle, and have a beneficial effect on cardiac function.
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Georgiev, David Georgiev. "Selective modification of biomolecules using radical mediated hydrothiolation chemistry." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31322.
Повний текст джерелаАнотація:
Intracellular protein-protein interactions (PPIs) play a vital role in many biological processes. Although they are viewed as of high biological interest they prove difficult to explore as potential targets for drug discovery. Numerous studies have shown α- helical peptides 'locked' in their respective bioactive structure can greatly increase their performance by increasing their target affinity, resistance to proteolysis as well as facilitating cellular uptake. A striking feature of literature to date is how few studies utilise different stapling techniques when developing inhibitors for PPIs. Current methods generally exploit ruthenium catalysed ring closing metathesis (RCM) or copper catalysed alkyne/azide click (CuAAC) chemistry to generate geometrically constrained peptides. Even though these methods have shown great potential they both share a fundamental limitation as the chemistry can only be employed on small synthetic peptides and cannot be extended to larger proteins. Thiol-ene coupling (TEC) chemistry (Chapter 1) which is often described as a 'click' reaction due to its fast reaction rates, high yields, wide functional group tolerance and insensitivity to ambient oxygen and water has the potential to solve this challenge. Thiol-ene chemistry was investigated as an alternative stapling strategy by employing the naturally occurring amino acid L-cysteine (Cys) as a source of the thiyl radical and L-homoallylglycine (Hag), a non-natural amino acid shown to act as a methionine surrogate in protein synthesis to act as a source of an alkene functionality to form a potentially expressible thioether tether in Chapter 2. However, due to unsatisfactory results from the intramolecular thiol-ene cyclisation at the molar concentrations required for peptide or protein modification, and a promising new lead, the closely related thiol-yne reaction was investigated as an alternative in Chapter 3. Using a small library of peptides (14 mers) derived from α-Synuclein (αSyn), a protein mainly found in the presynaptic terminals in the brain and is believed to be key to the pathological progress of Parkinson's disease, a successful macrocyclisation was achieved between the side chains of cysteine (Cys) and homopropargylglycine (Hpg). Although the vinyl-thioether tether did not confer any helical conformation on the stapled peptides, the results clearly demonstrate a potential route for the development of expressible staples. Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labelling (SDSL) of biomolecules has become a powerful tool for studying the structure and conformational dynamics of biomolecules. Typically, proteins are modified in a site-specific manner by utilising the side chains of cysteine residues to form disulphide bonds with spin active compounds, however, this strategy has its limitations. In Chapter 3 thiol-ene chemistry was investigated as an alternative biorthogonal method to spin label proteins and peptides. The newly synthesised sulfhydryl bearing nitroxide spin label was found to degrade upon exposure to radical promoting conditions, however, an alternative strategy was explored using more classical thiol-Michael chemistry to spin label dehydroalanine (Dha) modified peptides giving the desired spin labelled complex.
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HENNIG, PHILIPPE. "Etude par resonance magnetique nucleaire de l'interaction entre un beta bloquant, l'alprenolol et un fragment d'anticorps monoclonal, le peptide a13g." Paris 6, 1992. http://www.theses.fr/1992PA066182.
Повний текст джерелаАнотація:
L'etude par resonance magnetique nucleaire de ce sujet s'est effectuee en plusieurs etapes. Tout d'abord, il a ete indispensable d'obtenir une attribution complete des signaux rmn #1h, #1#3c, #1#5n du peptide a13g seul ou en presence d'alprenolol. Puis une etude de la conformation du peptide a13g, nous a permis d'apporter des elements indiquant une structure en boucle du peptide. Enfin, l'etude portant plus particulierement sur l'interaction entre les deux molecules, a fait intervenir plusieurs parametres. Les differences de deplacements chimiques, des mesures de temps de relaxation longitudinaux de certains signaux rmn de l'alprenolol, des noe intermoleculaire. L'ensemble des resultats a permis de determiner quelle region du peptide est impliquee dans l'interaction et quelle partie de l'alprenolol semble indispensable a cette interaction
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PRIGENT, YANN. "Structure en solution et en milieu micellaire d'un peptide antibiotique : la trichorzianine tavii. etude par resonance magnetique nucleaire et modelisation." Paris 6, 1992. http://www.theses.fr/1992PA066586.
Повний текст джерелаАнотація:
La trichorzianine tavii est un peptide antibiotique compose de 19 residus qui a la propriete de former, dans les bicouches phospholipides, ces canaux dont l'activite depend d'un potentiel trans-membranaire. Afin de tenter de comprendre le mecanisme d'action du peptide sur les membranes, l'etude structurale de tavii en solution dans le methanol et en milieu micellaire (rapport molaire sds/tavii=50) a ete realisee par resonance magnetique nucleaire #1h, #1#3c et #1#5n. Une attribution complete des signaux rmn a ete realisee pour ces trois noyaux. De nombreuses informations conformationnelles ont ete obtenues a l'aide d'experiences de rmn 2d (cosy-dqf, hohaha, noesy, roesy, hmqc, hm3c). Les valeurs de constantes de couplage 3jhn-ch, les coefficients de temperature des groupes nh et cc, les temps de relaxation longitudinaux #1#3c ont ete determines et les effets overhauser nucleaires analyses quantitativement. L'ensemble de ces resultats a permis de realiser la modelisation du peptide. Les conformations de la trichorzianine tavii en solution dans le methanol et en milieu micellaire, determinees par ces methodes, semblent similaires et sont caracterisees par une helice droite rigide, coudee au niveau des residus u#9-s#1#0 et incluant deux tours d'helice 3#1#0 au niveau de la proline et un tour mixte /3#1#0 en partie n-terminale. En presence de sds, tavii apparait aussi structure qu'en solution methanolique. Ces resultats suggerent que le mecanisme d'action de ce peptide sur les membranes met en jeu une reorientation globale de l'helice dans la bicouche et non un mouvement d'une partie du squelette peptidique
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Sani, Marc-Antoine. "Apoptosis Regulation via the Mitochondrial Pathway : Membrane Response upon Apoptotic Stimuli." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1883.
Повний текст джерелаАнотація:
The aim of this thesis was the investigation of the mitochondrial response mechanisms upon apoptotic stimuli. The specific objectives were the biophysical characterization of membrane dynamics and the specific roles of lipids in the context of apoptotic regulation occurring at the mitochondrion and its complex membrane systems. The BH4 domain is an anti-apoptotic specific domain of the Bcl-2 protein. Solid phase peptide synthesis was used to produce large amount of the peptide for biophysical studies. A protocol has been established and optimized, guarantying the required purity for biophysical studies. In detail the purification by high performance liquid chromatography and the characterisation via mass spectroscopy are described. The secondary structure of BH4 changes significantly in the presence of lipid vesicles as observed by infrared spectroscopy and circular dichroism. The BH4 peptide aggregates at the membrane surface and inserts slightly into the hydrophobic part of the membrane. Using nuclear magnetic resonance (NMR) and calorimetry techniques, it could even be shown that the BH4 domain modifies the dynamic and organization of the liposomes which mimic a mitochondrial surface. The second study was on the first helix of the pro-apoptotic protein Bax. This sequence called Bax-α1 has the function to address the cytosolic Bax protein to the mitochondrial membrane upon activation. Once again a protocol has been established for the synthesis and purification of this peptide. The aim was to elucidate the key role of cardiolipin, a mitochondria-specific phospholipid, in the interaction of Bax-α1 with the mitochondrial membrane system. The NMR and circular dichroism studies showed that Bax-α1 interacts with the membrane models only if they contain the cardiolipin, producing a strong electrostatic lock effect which is located at the membrane surface. Finally, a new NMR approach was developed which allows the investigation of the lipid response of isolated active mitochondria upon the presence of apoptotic stimuli. The goal was there to directly monitor lipid specific the occurring changes during these physiological activities.
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Nitin, Nitin. "Optical and MR Molecular Imaging Probes and Peptide-based Cellular Delivery for RNA Detection in Living Cells." Diss., Available online, Georgia Institute of Technology, 2005, 2005. http://etd.gatech.edu/theses/available/etd-08102005-120350/.
Повний текст джерелаАнотація:
Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2006.Dr. X. Hu, Committee Member ; Dr. Al Merrill, Committee Member ; Dr. Niren Murthy, Committee Member ; Dr. Gang Bao, Committee Chair ; Dr. Nicholas Hud, Committee Member. Includes bibliographical references.
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Cady, Sarah Diane. "Investigation of the structure and dynamics of the M2 transmembrane peptide of the influenza A virus by solid-state nuclear magnetic resonance." [Ames, Iowa : Iowa State University], 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3389091.
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Schoelzel, Daniel [Verfasser], та Gunnar [Gutachter] Schröder. "Structural characterization of recombinant and pathogenic Amyloidβ(1-42)-Peptide by solid-state Nuclear Magnetic Resonance Spectroscopy / Daniel Schoelzel ; Gutachter: Gunnar Schröder". Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2020. http://d-nb.info/1211813908/34.
Повний текст джерела
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Wahlström, Anna. "NMR studies on interactions between the amyloid β peptide and selected molecules". Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-60346.
Повний текст джерелаАнотація:
Alzheimer’s disease is an incurable neurodegenerative disorder linked to the amyloid β (Aβ) peptide, a 38-43 residue peptide. The detailed molecular disease mechanism(s) is (are) unknown, but oligomeric Aβ structures are proposed to be involved. In common for the papers in this thesis is interactions; interactions between Aβ(1-40) and selected molecules and metal ions. The purpose has been to find out more about the structural states that Aβ can adopt, in particular the β-sheet state, which probably is linked to the oligomeric structures. The methods used have been nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence spectroscopy using Thioflavin T (ThT). Upon addition of SDS/LiDS detergent or Congo red (CR) to Aβ(1-40), the initial random coil/PII-helix state was transformed into β-sheet and, in the case of detergent, a final α-helical state. In contrast to SDS/LiDS and CR, the dimeric Affibody molecule locks monomeric Aβ(1-40) in a β-hairpin state. It was found that by truncating the flexible N-terminal end of the Affibody molecule its affinity to Aβ was improved. The aggregation of Aβ(1-40) was further studied in the presence of a β-cyclodextrin dimer by a kinetic assay using ThT. Although having a weak dissociation constant in the millimolar range, the β-cyclodextrin dimer modified the aggregation pathways of Aβ. Finally Aβ(1-40) was studied in presence of Cu2+ and Zn2+ at physiological and low pH. Cu2+ was observed to maintain its specific binding to Aβ when decreasing the pH to 5.5 while Zn2+ behaved differently. This could be of importance in the Alzheimer’s disease brain in which the environment can become acidic due to inflammation. In conclusion the results show that Aβ(1-40) is very sensitive to its environment, responding by adopting different conformations and aggregating in aqueous solutions. The β-sheet state is induced by varying molecules with different properties, properties that govern the final Aβ state.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.
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Herrera, Alvaro Ivan. "Evaluation of NMR structural studies on a family of membrane active channel forming peptides." Diss., Kansas State University, 2015. http://hdl.handle.net/2097/19164.
Повний текст джерелаАнотація:
Doctor of PhilosophyBiochemistry and Molecular Biophysics
Om Prakash
John M. Tomich
As part of the ongoing development of a channel forming peptide with the potential to be used clinically to treat cystic fibrosis, a number of structural studies using solution NMR spectroscopy have been carried out on a number of the test sequences. Given their structural similarities of the monomers it is important to evaluate whether or not there is a compelling need to determine the solution NMR structure of next-generation peptides. The determination of the NMR monomeric solution structure of peptides NK₄-M2GlyR-p22 and NK₄-M2GlyR-p20 T17R S20W in TFE solution and SDS micelles sample shows predominantly alpha-helical conformations for both sequences with an extended conformation for the N-terminal lysine residues. The I[subscript max], K[subscript 1/2] and Hill coefficient, derived from data on ion conductance across monolayers of MDCK cells, were used to compare the ion conductance properties of the peptide sequences. Peptide NK₄ M2GlyR p20 T17R S20W has both a higher I[subscript MAX] (43.8 ± 2.8 μA/cm²) and a lower K[subscript 1/2] (58 ± 8 μM) compared to other M2GlyR derived peptides with calculated NMR structures. All available molecular structures calculated by NMR for M2GlyR derived peptides were compared and the correlation of the structural changes observed in the NMR structures with the ion conductance changes was evaluated. The NMR structures were found to have limited predicting potential over the ion conduction data. NMR determined structures have provided an experimentally based starting point for studies of the channels formed by the family of M2GlyR peptides. Computer simulations account for inter peptide interactions and packing effects that are not experienced by the monomeric form of the peptides in the NMR samples that have been used until now. The determination of the structure of the oligomeric peptide channels is deemed needed to improve the relevance of future use of NMR in this project. The use of larger membrane mimicking agents, isotopically labeled (¹⁵N, ¹³C) samples, 3D NMR experiments and potentially solid state NMR would be required to accomplish that task.
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Höger, Geralin. "Self-Organization of β-Peptide Nucleic Acid Helices for Membrane Scaffolding". Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2019. http://hdl.handle.net/21.11130/00-1735-0000-0003-C187-A.
Повний текст джерела
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Basi, Reddy Sreenivasulu Reddy, and s3046678@student rmit edu au. "A novel gold nanoparticle-based approach for the rapid diagnosis of meningococcal infection." RMIT University. Applied Sciences, 2008. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080730.165053.
Повний текст джерелаАнотація:
The bacterial meningitis caused by Neisseria meningitidis is responsible for considerable morbidity and mortality throughout the world. Given the limitations of existing diagnostic tests and the severity of the illness associated with the disease, there is a clear requirement for a rapid and specific diagnostic assay. This thesis describes the development of nanoparticle based tests for the detection of Neisseria meningitidis specific cell surface markers. As an initial target antigen, a recombinant form of highly conserved outer membrane protein, OMP85 was used. Within the OMP85 protein sequence, a predicted antigenic sequence between residues 720 and 745 was identified and found to be unique to this organism. This amino acid sequence was synthesised as peptide (SR1) with a gly-gly-cysteine spacer sequence at the N-terminus using t-boc chemistry. Also, the major virulence factor, capsular polysaccharide of N. meningitidis serogroup B bacteria was purified. Polyclonal antibodies were raised against purified OMP85 antigen in rabbits and against SR1 peptide and also against formalin inactivated N. meningitidis serogroup B whole cell bacteria in sheep. This panel of different antibodies including the commercial anti-capsular monoclonal antibodies were examined for cross reactivity against a range of closely related Gram negative bacteria. Based on these cross-reactivity studies, a highly specific anti-NM antibody was developed following purification of the anti-SR1 antiserum by immuno-affinity chromatography. Purified OMP85 antigen and anti-OMP85 antibody were successfully conjugated on 13, 30, 40, 50 and 60 nm gold nanoparticles by an electrostatic adsorption method. Coupling of the gold nanoparticles results in a shift of the respective surface plasmon peak toward longer wavelengths (in the range of 600-800 nm) resulting in a change of the colour of the colloidal suspension from red to purple to blue. An attempt was made to develop a rapid diagnostic assay based on gold nanoparticle induced colour shift assay for N. meningitidis by utilising the specific interaction of OMP85 and anti-OMP85 antibody conjugated to gold nanoparticles as a model system. However, this system was not reproducible and is likely to be due to problems with stability of gold nanoparticles during the conjugation process. As an alternative approach, a highly selective quartz crystal microbalance (QCM)-based immunosensor was designed using the same OMP85/anti-OMP85 antibody system. A method was developed using polyvinylidene fluoride (PVDF) coated QCM crystals with protein A for the directional orientation of the antibodies. To further enhance the sensitivity of the test, OMP85-conjugated gold nanoparticles were used as signal amplification probes for the reproducible detection of the target down to 300 ng/mL, corresponding to a five fold increase in sensitivity compared to detection of OMP85 antigen alone. Also, this sensor has successfully been employed to detect whole cell bacteria at a concentration as low as 100 cfu/mL. Thus, in this study using the real-time QCM measurements, a novel strategy has been developed for the sensitive detection of both N. meningitidis bacteria and the protein antigen at very low concentrations, using gold nanoparticles as signal amplification probes.
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Salza, Romain. "Les réseaux d’interactions de l’endostatine, de l’angiogenèse à la maladie d’Alzheimer." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10119.
Повний текст джерелаАнотація:
La matrice extracellulaire est composée d’environ 300 protéines et protéoglycanes qui constituent le matrisome et de 800 protéines associées (Naba et al., 2012a) et glycosaminoglycanes. C’est un protéome sous-exploré qui est modifié dans de nombreuses pathologies. Les fragments bioactifs issus de la matrice extracellulaire (matricryptines) sont capables de réguler des processus physiopathologiques et notamment l’angiogenèse et les pathologies cérébrales (Ricard-Blum and Salza, 2014). Environ 90 % des patients atteints de la maladie d’Alzheimer (MA) ont une angiopathie amyloïde cérébrale. L’angiogenèse contribue au déroulement de la MA. Nous nous sommes intéressés à l’endostatine (ES), une matricryptine du collagène XVIII qui possède des activités anti-angiogéniques, anti-tumorales et est également présente dans les plaques amyloïdes chez les patients atteints de la MA. Elle est libérée par les neurones et est capable de former des fibrilles amyloïdes in vitro (Kranenburg et al., 2003). Elle pourrait donc avoir une implication dans la MA. Nous avons montré que l'ES est présente dans le liquide céphalorachidien et que le rapport de sa concentration à celle des marqueurs classiques de la MA permet d’améliorer le diagnostic des patients atteint de démence fronto-temporale (DFT) et de discriminer les patients atteints de MA de ceux atteint de DFT et de pathologie nonMA/nonDFT. Nous avons établi les répertoires d’interactions extracellulaire du peptide -amyloïde (1-42) sous formes monomérique, oligomérique, fibrillaire ou agrégée et montré que l’oligomérisation et la fibrillogenèse augmentent la capacité d’interaction du peptide -amyloïde. Nous avons établi le réseau d’interaction global de l’endostatine par résonance plasmonique de surface en mode imagerie et identifiés 21 nouveaux partenaires de cette matricryptine. Nous avons plus particulièrement caractérisé son interaction avec la Procollagen C-Proteinase Enhancer-1, une protéine dont nous avons montré qu’elle donne naissance à une matricryptine anti-angiogénique. Nous avons enfin construit les réseaux d’interactions extracellulaires spécifiques de l’angiogenèse et de la maladie d’Alzheimer et des processus amyloïdes pour identifier les protéines connectant ces deux processus qui sont des cibles thérapeutiques potentielles. Ces réseaux d’interactions ont été créés à l’aide de 239 interactions que nous avons identifiées expérimentalement et des interactions décrites dans la littérature. Ces données seront à terme disponibles dans la base de données spécifique des interactions extracellulaires créée au laboratoire, MatrixDB, dans la nouvelle version à laquelle nous avons contribuéThe extracellular matrix include approximately 300 proteins and proteoglycans which constitute the matrisome and 800 associated proteins (Naba et al., 2012a) and glycosaminoglycans. It is an under-explored proteome which is modified in many diseases. Extracellular matrix bioactives fragments (matricryptins) are able to regulate physiopathological process like angiogenesis and cerebral disorders (Ricard-Blum and Salza, 2014). About 90 % of patients with Alzheimer's disease (AD) have cerebral amyloid angiopathy. Angiogenesis contributes to the development of AD. We are studying endostatin (ES), a matricryptin of collagen XVIII which has anti-angiogenic and anti-tumoral activities and is also present in amyloid plaques in AD patients. ES is released by neurons and is able to form amyloid fibrils in vitro (Kranenburg et al., 2003). This anti-angiogenic matricryptin could therefore be involved in AD. We have shown that ES is present in the cerebrospinal fluid of AD patients and the ratio of its concentrations to conventional markers of AD improves the diagnosis of patients with frontotemporal dementia (FTD) and discriminate AD patients from those suffering from FTD and pathology noAD/noDFT. We have established the extracellular interactions repertoires of the -amyloid peptide (1-42) in monomeric, oligomeric, fibrillar or aggregated forms and showed that the oligomerization and fibrillogenesis increase the interaction capacity of the -amyloid peptide. We have established the global interaction network of endostatin by surface plasmon resonance imaging and identified 21 new partners of this matricryptin. Specifically, we characterized its interaction with the Procollagen C-Proteinase Enhancer-1, a protein which gives rise to an anti-angiogenic matricryptin. We finally built networks of specific extracellular interactions of angiogenesis and of Alzheimer's disease and amyloid process to identify proteins connecting these two processes that are potential therapeutic targets. These interaction networks have been built using 239 interactions including those we have identified experimentally and those described in the literature. This data will be available in the database specific of extracellular interactions created in the laboratory, MatrixDB, in the new version of which we contributed
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Junior, José Carlos Bozelli. "Interação do peptídeo de defesa do hospedeiro tritrpticina (TRP3) e seus análogos com membranas modelo: efeitos na estrutura e dinâmica da membrana." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-07122015-113945/.
Повний текст джерелаАнотація:
Tritrpticina (TRP3) é um peptídeo antimicrobiano com 13 resíduos de amino ácidos com três Ws sequenciais. Com o objetivo de contribuir para a compreensão de seu mecanismo de ação, realizaram-se estudos funcionais e conformacionais da TRP3 e de dois análogos onde um (WLW) ou dois (LWL) W foram substituídos por L. Os peptídeos foram igualmente ativos contra bactérias Gram positivas e negativas. Sua atividade hemolítica requereu concentrações maiores, diminuindo na ordem TRP3>WLW>LWL. Os peptídeos permeabilizaram membranas modelo de E. coli ou contendo fosfolipídios carregados negativamente. Espectros de CD sugeriram que os peptídeos adquirem diferentes conformações ao se ligarem a bicamadas e micelas. Estudos de fluorescência mostraram que a ligação a membranas decresce na ordem: TRP3>WLW>LWL e que os peptídeos se localizam próximos à interface membrana-água. Espectros de RPE de marcadores de spin lipídicos indicaram que a ligação dos peptídeos altera a organização dos lipídios, aumentando o empacotamento molecularTritrpticin (TRP3) is a 13-residue antimicrobial peptide that contains three sequential Ws. With the aim of contributing to the understanding of its mechanism of action, functional and conformational studies were performed with TRP3 and two of its analogues where one (WLW) or two (LWL) of the W were replaced by L. The peptides were equally active against both Gram positive and Gram negative bacteria. Higher concentrations were required for hemolytic activity which varied in the order: TRP3>WLW>LWL. The peptides permeabilized membranes model membranes mimicking E. coli\'s lipid composition or containing different negatively charged phospholipids. CD spectra suggested the peptides acquired different conformations upon binding to bilayers or micelles. Fluorescence studies showed that membrane binding decreases in the order: TRP3>WLW>LWL and that the peptides are located close to the water-membrane interface. EPR spectra of lipid spin labels indicated that peptide binding alter lipid organization, increasing molecular packing
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Doyen, Camille. "Utilisation de la RMN pour la caractérisation structurale et cinétique d'associations peptide-liposome comme aide à la conception de formulation." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASS110.
Повний текст джерелаАнотація:
L’encapsulation de principes actifs (PA) dans des liposomes est utilisée dans de nombreux domaines tels que les industries pharmaceutiques et cosmétiques. Les liposomes sont des systèmes d’administration de médicaments utilisés pour leur relargage contrôlé des PA. Pour augmenter l’efficacité de peptides comme PA, nous cherchons à optimiser des liposomes en jouant à la fois sur leur composition et la structure des peptides. Les paramètres à améliorer sont leurs interactions, l’efficacité d’encapsulation et la cinétique de relargage. Pour atteindre cet objectif, j’ai évalué l’intérêt de la spectroscopie par Résonance Magnétique Nucléaire (RMN) pour caractériser les peptides, les liposomes et leurs comportements pendant le relargage. Les peptides utilisés dans cette étude sont dérivés de l’apeline, qui a un intérêt pour la régulation homéostatique du système cardiovasculaire. Les structures des liposomes et des peptides ainsi que leurs interactions ont été étudiées par RMN ¹H et ³¹P et par cryo-EM. J’ai montré que les méthodes de diffusion par RMN, en s’appuyant sur la taille apparente des molécules, permettent de différencier les compartiments internes et externes des liposomes et de suivre les cinétiques in situ en temps réel de relargage de peptide, sans perturber le processus. Les cinétiques de relargage ont aussi été quantifiées par intégration spectrale de spectres RMN ¹H. J’ai aussi montré que la méthode de préparation des liposomes avait un impact sur leur structure, leur cinétique de relargage et leurs interactions avec des peptides. L’addition d’une chaîne lipidique augmente les interactions avec les liposomes, mais la longueur et le type de chaîne induisent peu de différences. Les outils de RMN mis en place pourront être étendus à d’autres PA et systèmes d’administration pour des applications pharmaceutiques et cosmétiquesThe encapsulation of active ingredients (AI) in liposomes is used in several domains such as pharmaceutical and cosmetic industries. Liposomes are drug delivery systems (DDS) used for a controlled release of AIs. To increase the efficiency of peptide drugs, I aim at designing optimized peptide/liposomes formulation by playing both on their composition and peptide structure. Potential parameters to be improved are their interactions, encapsulation efficiency and release kinetics. To reach this goal, I explored the potentiality of Nuclear Magnetic Resonance (NMR) spectroscopy to characterize peptides, liposomes and their behavior during release. The AIs used in this study are apelin-derived peptides that are of interest for homeostasis regulation of the cardiovascular system. Liposome and peptide structures as well as their interactions were characterized by ¹H and ³¹P NMR and cryo-EM. I showed that diffusion NMR methods that report on the apparent size of molecules can discriminate between the inner and the outer space of liposomes and for release quantification in-situ and in real-time without perturbing the process. Moreover, ¹H NMR spectra were used to monitor and quantify peptide release kinetics by spectral integration. I showed that the preparation method of liposomes drastically impacts their structure, release kinetics and interactions with peptides. Addition of a lipid chain increases the interaction with the liposomes, but the type and length of the chain induce only few differences. This approach could certainly be extended to other AIs and DDSs used for pharmaceutical and cosmetic applications
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Sani, Marc Antoine. "Apoptosis regulation via the mitochondrial pathway : membrane response upon apoptotic stimuli." Thesis, Bordeaux 1, 2008. http://www.theses.fr/2008BOR13651/document.
Повний текст джерелаАнотація:
Le but de cette thèse est de montrer la réponse de la membrane mitochondriale au cours la régulation de l’apoptose en étudiant l’effet de domaines clés sur la dynamique membranaire et l’importance de la composition phospholipidiques des modèles utilisés. Le domaine BH4 est la partie spécifique anti-apoptotique de la famille Bcl-2. La première étape a été de synthétiser le peptide par voie chimique en utilisant la synthèse peptidique en phase solide. Un protocole décrivant les étapes de purification par chromatographie liquide et de caractérisation par spectroscopie de masse, garantissant une pureté indispensable pour des études biophysiques, a été établi. La modification de la structure secondaire du peptide interagissant avec des vésicules a été étudiée par spectroscopie infrarouge ainsi que par dichroïsme circulaire. Le peptide s’agrège à la surface et s’insère peu profondément dans la partie hydrophobe de la membrane. En utilisant la résonance magnétique nucléaire (RMN) et la calorimétrie, il a été montré que le peptide BH4 modifie l’organisation et la dynamique des liposomes mimant la surface mitochondriale. La deuxième étude a porté sur la première hélice de la protéine pro-apoptotique Bax (Bax-a1) qui a la propriété de diriger la protéine cytosolique vers la mitochondrie. Un protocole de synthèse et purification a été à nouveau établi. Le but de cette étude est de démontrer le rôle de l’interaction spécifique entre la cardiolipine, un phospholipide uniquement présent dans la mitochondrie et le peptide Bax-a1. Les études RMN ont montré que Bax-a1 n’interagissait uniquement que si la cardiolipine était présente, produisant un fort effet électrostatique piégeant le peptide à la surface de la membrane. Enfin, un nouveau protocole permettant d’étudier la réponse des lipides de mitochondries isolées toujours actives par RMN est présenté. Le but est de pouvoir directement observer les modifications subies par chaque phospholipide de la mitochondrie.The aim of this thesis was the investigation of the mitochondrial response mechanisms upon apoptotic stimuli. The specific objectives were the biophysical characterization of membrane dynamics and the specific roles of lipids in the context of apoptotic regulation occurring at the mitochondrion and its complex membrane systems. The BH4 domain is an anti-apoptotic specific domain of the Bcl-2 protein. Solid phase peptide synthesis was used to produce large amount of the peptide for biophysical studies. A protocol has been established and optimized, guarantying the required purity for biophysical studies. In detail the purification by high performance liquid chromatography and the characterisation via mass spectroscopy are described. The secondary structure of BH4 changes significantly in the presence of lipid vesicles as observed by infrared spectroscopy and circular dichroism. The BH4 peptide aggregates at the membrane surface and inserts slightly into the hydrophobic part of the membrane. Using nuclear magnetic resonance (NMR) and calorimetry techniques, it could even be shown that the BH4 domain modifies the dynamic and organization of the liposomes which mimic a mitochondrial surface. The second study was on the first helix of the pro-apoptotic protein Bax. This sequence called Bax-a1 has the function to address the cytosolic Bax protein to the mitochondrial membrane upon activation. Once again a protocol has been established for the synthesis and purification of this peptide. The aim was to elucidate the key role of cardiolipin, a mitochondria-specific phospholipid, in the interaction of Bax-a1 with the mitochondrial membrane system. The NMR and circular dichroism studies showed that Bax-a1 interacts with the membrane models only if they contain the cardiolipin, producing a strong electrostatic lock effect which is located at the membrane surface. Finally, a new NMR approach was developed which allows the investigation of the lipid response of isolated active mitochondria upon the presence of apoptotic stimuli. The goal was there to directly monitor lipid specific the occurring changes during these physiological activities
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Nilsson, Jonas. "Design, Synthesis and Characterization of Small Molecule Inhibitors and Small Molecule : Peptide Conjugates as Protein Actors." Doctoral thesis, Linköpings universitet, Organisk Kemi, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-3943.
Повний текст джерелаАнотація:
This thesis describes different aspects of protein interactions. Initially the function of peptides and their conjugates with small molecule inhibitors on the surface of Human Carbonic Anhydrase isoenzyme II (HCAII) is evaluated. The affinities for HCAII of the flexible, synthetic helix-loop-helix motif conjugated with a series of spacered inhibitors were measured by fluorescence spectroscopy and found in the best cases to be in the low nM range. Dissociation constants show considerable dependence on linker length and vary from 3000 nM for the shortest spacer to 40 nM for the longest with a minimum of 5 nM for a spacer with an intermediate length. A rationale for binding differences based on cooperativity is presented and supported by affinities as determined by fluorescence spectroscopy. Heteronuclear Single Quantum Correlation Nuclear Magnetic Resonance (HSQC) spectroscopic experiments with 15N-labeled HCAII were used for the determination of the site of interaction. The influence of peptide charge and hydrophobicity was evaluated by surface plasmon resonance experiments. Hydrophobic sidechain branching and, more pronounced, peptide charge was demonstrated to modulate peptide – HCAII binding interactions in a cooperative manner, with affinities spanning almost two orders of magnitude. Detailed synthesis of small molecule inhibitors in a general lead discovery library as well as a targeted library for inhibition of α-thrombin is described. For the lead discovery library 160 members emanate from two N4-aryl-piperazine-2-carboxylic acid scaffolds derivatized in two dimensions employing a combinatorial approach on solid support. The targeted library was based on peptidomimetics of the D-Phe-Pro-Arg showing the scaffolds cyclopropane-1R,2R-dicarboxylic acid and (4-amino-3-oxo-morpholin-2-yl)- acetic acid as proline isosters. Employing 4-aminomethyl-benzamidine as arginine mimic and different hydrophobic amines and electrophiles as D-phenylalanine mimics resulted in 34 compounds showing IC50 values for α-thrombin ranging more than three orders of magnitude with the best inhibitor showing an IC50 of 130 nM. Interestingly, the best inhibitors showed reversed stereochemistry in comparison with a previously reported series employing a 3-oxo-morpholin-2-yl-acetic acid scaffold.
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Ramström, Margareta. "Analysis of Complex Biological Samples using Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry." Doctoral thesis, Uppsala University, Analytical Chemistry, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5729.
Повний текст джерелаАнотація:
Studies of protein and peptide expression are vital in order to understand complex biological systems. As demonstrated in this thesis, on-line packed capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS) is a useful analytical tool for such studies.
A proteomics method, based on global tryptic digestion and subsequent separation and detection of the peptides by LC-FTICR MS, was developed for qualitative analysis of body fluids. Initial experiments on cerebrospinal fluid (CSF) provided results that were comparable or superior to those achieved by more time- and sample-consuming techniques. The method was also successfully applied on plasma and amniotic fluid. One of the major challenges in proteomics is the broad dynamic range of proteins in biological matrices. The advantages of removing high-abundant components from CSF and plasma prior to MS were demonstrated.
In order to search for potential biomarkers, mass chromatograms of CSF from patients suffering from amyotrophic lateral sclerosis (ALS) and controls were compared using an in-house constructed pattern recognition program. ALS-specific patterns were observed, and four out of five unknown samples were correctly assigned. Alternative strategies to quantitatively compare two pools of samples rely on differential chemical labeling. The performance of one such method, quantification-using-enhanced-signal-tags, was investigated in complex sample analysis. The experimental intensity ratios were proven to be consistent with the prepared concentration ratios of abundant proteins in CSF.
Finally, the thesis reports on the first experiments where electron capture dissociation (ECD) was successfully incorporated in on-line LC-MS experiments. ECD and nozzle-skimmer fragmentation were applied to a sample of endocrine peptides extracted from mouse pancreatic islets. The two fragmentation methods provided complementary information. However, the method needs further optimization before it can be applied in the analysis of more complex samples, such as body fluids.
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Zhang, Liwen. "Characterization of histone post-translational modification using reversed-phase high performance liquid chromatography and fourier transform ion cyclotron resonance mass spectrometry." Connect to this title online, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1054660495.
Повний текст джерелаАнотація:
Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xv, 219 p.; also includes graphics (some col.) Includes bibliographical references (p. 147-173). Available online via OhioLINK's ETD Center
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Cornille, Fabrice. "Etude des interactions entre peptides et molécules de classe I du complexe majeur d'histocompatibilité, synthèse et étude physicochimique de la protéine de nucléocapside NCp10 du rétrovirus MoMuLU." Paris 5, 1991. http://www.theses.fr/1991PA05P604.
Повний текст джерела
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Kellenberger, Esther. "Determination par resonance magnetique nucleaire de la structure tridimensionnelle de petites proteines : _ peptide chimere stabilise par des ponts disulfure _ domaine carboxy-terminal de la sous-unite p44 du facteur de transcription humain tfiih." Université Louis Pasteur (Strasbourg) (1971-2008), 2000. http://www.theses.fr/2000STR13033.
Повний текст джерелаАнотація:
La premiere partie traite d'ingenierie des proteines. Une sequence de type rgd a ete introduite dans une toxine de scorpion contenant trois ponts disulfure. La toxine chimere possede des proprietes antiagregantes. Sa structure tridimensionnelle revele qu'une helice alpha peut mimer un coude beta de type ii. Une deuxieme partie s'interesse aux proteines qui lient le zinc et qui sont impliquees dans la transcription des genes de classes ii chez les eucaryotes. _ une revue bibliographique decrit les proprietes du zinc et les differents motifs structuraux de liaison au zinc. _ un chapitre est consacre a la purification de la sous-unite hrpb10alpha de l'arn polymerase ii humaine. Cette sous-unite de 58 acides amines lie un ion zn 2 +. Le comportement biochimique ainsi que les premiers spectres enregistres sur la proteine purifiee mettent en evidence une tendance a l'agregation. _ un chapitre presente la determination de la structure tridimensionnelle du domaine carboxy-terminal de la sous-unite p44 (p44 3 2 1 - 3 9 5) de tfiih. Tfiih est un facteur de base de la transcription. Il est aussi un acteur essentiel de la reparation de l'adn par excision de nucleotides. Le domaine p44 3 2 1 - 3 9 5 lie deux ions zn 2 +. L'etude structurale revele un feuillet beta antiparallele a trois brins associe a une helice alpha. Elle temoigne egalement d'une dynamique localisee autour de l'un des sites de liaison au zinc. Le motif de coordination des ions zinc de p44 3 2 1 - 3 9 5 est original, bien que la topologie du domaine soit commun a d'autres proteines de liaison au zinc. Le travail sur p44 a fait appel a deux techniques propres a la resonance magnetique nucleaire des macromolecules biologiques : (1) le marquage isotopique et (2) l'automatisation de l'attribution des pics de correlation noes. L'utilisation et l'evaluation de ces techniques sont detaillees dans une partie methodologie.
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Grau, Campistany Ariadna. "Design, synthesis and study of the biological and biophysical activity of antimicrobial peptides." Doctoral thesis, Universitat de Barcelona, 2014. http://hdl.handle.net/10803/279307.
Повний текст джерелаАнотація:
The global emergence and spread of multidrug-resistant bacteria is an important public health issue. However, the antimicrobial pipeline remains unacceptably lean, in fact over the last 25 years, the number of antimicrobial agents that reach the market has sharply decreased. In this context, there is now a renewed interest in the search for drugs that have more than one target on the bacterial cell, rather than a specific chiral receptor or enzyme. Antimicrobial peptides (AMPs) are a class of antibiotics that have attracted great interest in the last few years because they rarely spur the development of resistant organisms as their mechanism of action involves disruption of the bacterial membrane.
In this thesis we report the design, preparation and activity of new compounds based on the sequence of the polymyxins, a class of antibiotics highly active against Gramnegative bacteria, and used clinically as last resort treatment for multidrug-resistant pathogens. The compounds synthesized proved to be highly active against both Grampositive and Gram-negative bacteria, including several strains of resistant bacteria. Furthermore, biophysical experiments using liposomes and monolayers as model membranes and flow cytometry and transmission electron microscopy using bacteria were carried out to study the mechanism of action of these compounds. The results of the most active candidate indicate that an alternative, non-membrane dependent mechanism of action might be involved.
In the second part of the thesis, a series of 9 peptides were designed and synthesized from repeated KIAGKIA motifs, based in the sequence of the antimicrobial peptide PGLa from the magainin family, with lengths between 14 and 28 amino acids. Circular dichroism spectroscopy showed that they all formed alpha-helices when found in a lipid environment. Biological assays for haemolysis and antimicrobial activity, as well as fluorescence vesicle leakage and solid-state NMR spectroscopy, were used to correlate peptide length with membrane activity. These data are fully consistent with the formation of transmembrane pores. Only peptides that are long enough to span the hydrophobic bilayer core can induce vesicle leakage, haemolysis, and inhibit bacterial growth. The shorter peptides do not show these effects. Solid-state NMR analysis in oriented bilayers with different thickness also demonstrated the need for a minimum peptide length to flip from a surface-bound alignment into a more inserted, possibly even transmembrane state. With increasing length the peptides start to tilt and perturb the bilayer. Since the threshold behaviour seen for biological activity closely matches the biophysical results, the peptides could be used as molecular rulers to determine the thickness of bacterial membranes, showing that E. coli (≈ 27 Å) < S. aureus and P. aeruginosa (≈ 30 Å) < E. faecalis (≈ 34 Å).L'aparició i propagació mundial de bacteris resistents a múltiples fàrmacs s’ha convertit en un problema clínic molt important. No obstant això, el nombre de nous antimicrobians que es troben en les últimes etapes de desenvolupament és molt baix. Els pèptids antimicrobians són una classe d’antibiòtics que han despertat gran interès en els últims anys pel fet que poques vegades estimulen el desenvolupament d’organismes genèticament resistents ja que la seva diana terapèutica és principalment la membrana bacteriana. En aquesta tesi es descriu el disseny, preparació i activitat de nous compostos basats en la seqüència de les polimixines, un tipus de pèptids antimicrobians altament potents contra bacteris Gram-negatius i usats clínicament. Els compostos sintetitzats van presentar elevada activitat tant en bacteris Gram positius com Gram-negatius, incloent diverses soques resistents. Addicionalment, experiments biofísics usant liposomes i monocapes com a models de membrana i citometria de flux i microscòpia electrònica de transmissió usant bacteris es van usar per estudiar el mecanisme d’acció d’aquests compostos. Els resultats del candidat més actiu semblen indicar que presenta un mecanisme d’acció alternatiu on la membrana bacteriana no és l’única diana terapèutica. En la segona part de la tesi, es descriu el disseny i síntesi d’una sèrie de 9 pèptids, basats en la seqüència del pèptid antimicrobià PGLa de la família de les magainines. Els pèptids alfa-helicoïdals (vist mitjançant l’ús de dicroïsme circular), presenten repeticions del heptàmer KIAGKIA, amb llargades de 14 a 28 aminoàcids. Assaigs biològics, de fluorescència i de RMN de fases condensades es van usar per relacionar la llargada peptídica amb la activitat en la membrana. Les dades obtingudes són consistents amb la formació de porus transmembrana, només aquells pèptids prou llargs per travessar la bicapa lipídica indueixen permeabilització, hemòlisi i inhibeixen el creixement bacterià mentre que els pèptids curts no mostren aquests efectes. L’anàlisi usant RMN de fases condensades amb lípids de diferent llargada també va mostrar la necessitat d’una llargada mínima dels pèptids per passar d’un estat superficial a un estat més inserit, probablement transmembrana. Atès que els llindars observats per a l'activitat biològica coincideixen estretament amb els resultats biofísics, els pèptids van ser utilitzats com a regles moleculars per determinar el gruix de les membranes bacterianes, és a dir E. coli (≈ 27 Å) < S. aureus i P. aeruginosa (≈ 30 Å) < E. faecalis (≈ 34 Å).
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Dijkman, Patricia M. "Biophysical studies of membrane protein structure and function." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:ad0fde85-c4b6-48a1-b51b-d304aca45402.
Повний текст джерелаАнотація:
Membrane proteins play a key role in numerous physiological processes such as transport, energy transduction in respiratory and photosynthetic systems, and signal transduction, and are of great pharmaceutical interest, comprising more than 60% of known drug targets. However, crystallisation of membrane proteins, and G protein-coupled receptors (GPCRs) in particular, still relies heavily on the use of protein engineering strategies, which have been shown to hamper protein activity. Here, a range of biophysical methods were used to study the structure and function of two membrane proteins, a prokaryotic peptide transporter, PepTSo and a GPCR, neurotensin receptor 1 (NTS1), using different membrane reconstitution methods to study the proteins in a native-like environment. Firstly, using the pulsed electron paramagnetic resonance (EPR) method of double electron-electron resonance (DEER) the conformation of PepTSo reconstituted into lipid bilayers was assessed and compared to previous structural data obtained from crystallography and modelling. The influence of the membrane potential and the presence of substrate on the conformational heterogeneity of this proton-coupled transporter were investigated. Secondly, NTS1 purification was optimized for biophysical study. Cysteine mutants were created and a labelling protocol was developed and optimized for fluorophore and nitroxide labelling studies. NTS1 was then studied by continuous-wave EPR, to assess the influence of ligand on local protein dynamics, and to assess the structure of a receptor segment known as helix 8, that was proposed to be an α-helix, but was only observed to be helical in one of the NTS1 crystallographic studies. Ensemble and single-molecule Förster resonance energy transfer (FRET), and DEER were combined to study the dimerisation behaviour of NTS1, showing novel dynamics of the interfacial associations. Finally, the signalling mechanism of NTS1 was also investigated using microscale thermophoresis (MST) to assess the affinity of the receptor for G protein in vitro in the absence of ligand, or in the presence of agonist or antagonist. MST measurements were performed in detergent and in nanodiscs of different lipid compositions, to assess the influence of the lipid environment on receptor function. In summary, this thesis demonstrates the potential of biophysical techniques to study various aspects of membrane protein structure and function in native-like lipid systems, complementing e.g. structural data obtained from crystallographic studies with functional data for membrane proteins in more native environments, as well as shedding light on protein dynamics. The work presented here provides novel insights into PepTSo transport, and in particular into NTS1 structure, signalling, and oligomerisation, opening up several avenues for future research.
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Sarrouj, Hiba. "DNP/solid state NMR probehead for the investigation of oriented membranes." Phd thesis, Université de Strasbourg, 2014. http://tel.archives-ouvertes.fr/tel-01038015.
Повний текст джерелаАнотація:
Helical membrane proteins comprise one third of the expressed proteins encoded in a typical genome. Other membrane proteins are typically beta sheets. Their function varies from pore formation, signaling to antimicrobial activity. They are also capable of transporting large cargo such as proteins or nucleic acids across the cell membrane. Recently, peptides have emerged as promising tools in drug delivery. Membrane proteins can be synthesized chemically or expressed and isotopically labeled in bacteria, isolated, purified and reconstituted into fully hydrated lipid bilayers. The bilayer orientation is kept mechanically by putting them between glass plates. While interacting with these bilayers they exhibit a variety of configurations depending on the lipids composition and thickness. Solid-state Nuclear Magnetic Resonance (NMR) on oriented bilayers is one way to access the topology of peptides associated with phospholipid membranes. Oriented membrane protein are difficult to study with analytical techniques because of their poor solubility outside the lipid membrane, difficulty of expression in bacteria in big quantities, difficulty to crystallize, and they are too large for solution NMR study. The intensity of an NMR signal depends on several factors such as polarization P and magnetic field magnitude B0. One of the major drawbacks of NMR spectroscopy is low sensitivity. This is caused by the small magnetic moment of the nuclear spins which results in a modest Zeeman splitting of the nuclear spin energy levels and therefore in a limited Boltzmann Polarization. The aim of this project is to obtain a better signal from membrane proteins. Thus a Low temperature (LT) solid state NMR with Dynamic Nuclear Polarization (DNP) probe head was created. DNP is an ingenious technique that is used to transfer polarization from highly polarized targets to less polarized nuclei using microwave irradiation. Microwaves will excite selectively the electron spins which will transfer their polarization to the pool of proton nuclei, the proton NMR signal can be enhanced by 660 times. A probe head for DNP enhanced solid state NMR at 100 K and 9.4 T is described. A probe head includes the mechanical piece that holds the sample in the magnetic center of the NMR magnet. It is a tunable antenna that irradiates and detects the rf fields used in NMR. The centerpiece of the probe is the solenoidal or saddle coil surrounding the sample. The feasibility of such a DNP experiment is proven on magic angle oriented sample spinning. These experiments are conducted on oriented samples wrapped into a rotor. Through their orientation with regards to B0 is lost, enhancement values as high as 17 are obtained. [...]
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Rost, Ulrike. "Organisation and Recognition of Artificial Transmembrane Peptides." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2016. http://hdl.handle.net/11858/00-1735-0000-002B-7CA6-D.
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Quinn, Steven D. "Advanced optical techniques to study biomolecular aggregation processes." Thesis, University of St Andrews, 2014. http://hdl.handle.net/10023/6137.
Повний текст джерелаАнотація:
Alzheimer's disease (AD) is characterised by a series of biomolecular aggregation events, which include the formation of neurotoxic protein structures composed of the β-amyloid (Aβ) peptide. In this thesis, fluorescence self-quenching (FSQ) between fluorescently-labelled peptides is introduced as a strategy for detecting and characterizing Aβ aggregates in solution, and for overcoming limitations associated with conventional methods. Using a combination of steady-state, picosecond time-resolved fluorescence and transmission electron microscopy, the fluorescence response of HiLyte Fluor 555-labelled Aβ peptides is characterised to demonstrate that Aβ self-assembly organizes the covalently attached probes in close proximity to trigger the self-quenching sensing process over a broad range of conditions. Importantly, N-terminal tagging of β-amyloid peptides is shown to not alter the self-assembly kinetics or the resulting aggregated structures. When performed in Förster resonance energy transfer (FRET) format, this method becomes a ratiometric platform to gain insights into amyloid structure and for standardizing in vitro studies of amyloid self-assembly. The ability of FSQ-based methods to monitor the inhibition of Aβ aggregation by model test compounds including the small heat shock protein (Hsp), the amyloid-binding alcohol dehydrogenase protein (ABAD) and bovine serum albumin (BSA) is also demonstrated. Given that Aβ is formed within the cell membrane and is known to induce its disruption, sophisticated single-molecule fluorescence spectroscopy methods were developed to quantify membrane dynamics induced by the presence of disrupting agents, such as Aβ and detergents. The solubilisation dynamics of single liposomes induced by the non-ionic surfactant Triton-X 100 (TX-100) were studied in real-time. Using this approach, the swelling and permeabilization steps of the solubilisation process were unambiguously separated within single FRET trajectories, and their kinetic details as a function of Triton-X 100 and presence of cholesterol within the membrane structure were examined. Finally, single-molecule stepwise-photobleaching techniques were employed to study the effect of Aβ oligomers interacting with supported-lipid bilayers, establishing a platform from which to investigate how the presence of a membrane layer affects Aβ oligomerization at the level of individual molecules. Overall, the fluorescence-based strategies for amyloid- and liposome-sensing presented in this work bridges the gap between current morphology-specific techniques and highly-specialized single-molecule methods to provide a biophysical toolbox to investigate the changes in structure, size and molecular interactions accompanying the amyloid aggregation pathway and for the screening of novel therapeutic and diagnostic agents.
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Oyler, Nathan Andrew. "SSNMR methods for determining structure in nucleosides and peptides /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/11589.
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LOPEZ, SOPHIE. "Resonance magnetique nucleaire et modelisation de triples helices et de peptides." Orléans, 1993. http://www.theses.fr/1993ORLE2023.
Повний текст джерелаАнотація:
Nous avons applique les techniques de resonance magnetique nucleaire et de modelisation moleculaire a deux types de composes biologiques. Dans un premier temps, nous avons etudie le complexe forme entre une double helice d'adn (un brin homopurine et un brin homopyrimidine de neuf paires de bases) et un troisieme brin de sept bases. La formation de cette triple helice est a la base d'une recente strategie therapeutique, puisqu'elle peut permettre de bloquer ou de reguler selectivement l'expression de genes par exemple. Avec l'aide des experiences de resonance magnetique du proton (cosy, tocsy, noesy) nous avons attribue la totalite des protons de la double helice et certains protons echangeables de la triple helice. Dans un deuxieme temps, nous nous sommes interesses a un peptide de onze acides amines, qui reproduit le site antigenique a de la proteine de surface du virus influenze. Ce peptide induit une protection contre le virus lorsqu'il est injecte a des souris. Nous avons extrait des spectres d'experiences bidimensionnelles des donnees, telles que les constantes de couplage et les intensites noe qui, converties en parametres structuraux (angles diedres, intervalles de distances), ont ete introduites comme contraintes experimentales pour generer des structures avec le logiciel de geometrie de distances disman. Des 150 modeles obtenus, nous en avons retenu trois selon divers criteres, pour effectuer des simulations de dynamique moleculaire. L'etude des trajectoires permet d'obtenir une image de la flexibilite des modeles sur une periode de 40 picosecondes
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Ahmed, Zareen. "Magnetic resonance spectroscopy of phospholamban and its interaction with Ca'2'+-ATPase." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343301.
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Fesinmeyer, Robert Matthew. "Chemical shifts define the structure and folding thermodynamics of polypeptides /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/11621.
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