Academic literature on the topic 'NMR, Paramagnetism, Protein Characterization'
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Journal articles on the topic "NMR, Paramagnetism, Protein Characterization"
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Arnesano, Fabio, Lucia Banci, and Mario Piccioli. "NMR structures of paramagnetic metalloproteins." Quarterly Reviews of Biophysics 38, no. 2 (May 2005): 167–219. http://dx.doi.org/10.1017/s0033583506004161.
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1. Introduction 1681.1 Genomic annotation of metalloproteins 1681.2 Why NMR structures? 1681.3 Why paramagnetic metalloproteins? 1692. General theory 1702.1 Nuclear and electron spins 1702.2 Hyperfine coupling 1712.3 The effect of the hyperfine coupling on the NMR shift: the hyperfine shift 1732.4 The effect of the hyperfine coupling on nuclear relaxation 1742.5 Interplay between electron spin properties and features of the NMR spectra 1783. Paramagnetism-based structural restraints 1803.1 Contact shifts and relaxation rates as restraints 1813.2 Locating the metal ion within the protein frame: pseudocontact shifts 1843.3 Cross-correlation rates 1863.4 Residual dipolar couplings 1883.5 Interplay between different restraints 1904. NMR without1H detection 1914.1 The protocol for 13C-detected protonless assignment of backbone and side-chains 1944.2 Protonless heteronuclear NMR experiments tailored to paramagnetic systems 1965. The use of lanthanides as paramagnetic probes 1986. The case of Cu(II) proteins 2027. Perspectives 2088. Acknowledgments 2099. References 209Metalloproteins represent a large share of the proteome and many of them contain paramagnetic metal ions. The knowledge, at atomic resolution, of their structure in solution is important to understand processes in which they are involved, such as electron transfer mechanisms, enzymatic reactions, metal homeostasis and metal trafficking, as well as interactions with their partners. Formerly considered as unfeasible, the first structure in solution by nuclear magnetic resonance (NMR) of a paramagnetic protein was obtained in 1994. Methodological and instrumental advancements pursued over the last decade are such that NMR structure of paramagnetic proteins may be now routinely obtained. We focus here on approaches and problems related to the structure determination of paramagnetic proteins in solution through NMR spectroscopy. After a survey of the background theory, we show how the effects produced by the presence of a paramagnetic metal ion on the NMR parameters, which are in many cases deleterious for the detection of NMR spectra, can be overcome and turned into an additional source of structural restraints. We also briefly address features and perspectives given by the use of 13C-detected protonless NMR spectroscopy for proteins in solution. The structural information obtained through the exploitation of a paramagnetic center are discussed for some Cu2+-binding proteins and for Ca2+-binding proteins, where the replacement of a diamagnetic metal ion with suitable paramagnetic metal ions suggests novel approaches to the structural characterization of proteins containing diamagnetic and NMR-silent metal ions.
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Piccioli, Mario. "Paramagnetic NMR Spectroscopy Is a Tool to Address Reactivity, Structure, and Protein–Protein Interactions of Metalloproteins: The Case of Iron–Sulfur Proteins." Magnetochemistry 6, no. 4 (September 26, 2020): 46. http://dx.doi.org/10.3390/magnetochemistry6040046.
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The study of cellular machineries responsible for the iron–sulfur (Fe–S) cluster biogenesis has led to the identification of a large number of proteins, whose importance for life is documented by an increasing number of diseases linked to them. The labile nature of Fe–S clusters and the transient protein–protein interactions, occurring during the various steps of the maturation process, make their structural characterization in solution particularly difficult. Paramagnetic nuclear magnetic resonance (NMR) has been used for decades to characterize chemical composition, magnetic coupling, and the electronic structure of Fe–S clusters in proteins; it represents, therefore, a powerful tool to study the protein–protein interaction networks of proteins involving into iron–sulfur cluster biogenesis. The optimization of the various NMR experiments with respect to the hyperfine interaction will be summarized here in the form of a protocol; recently developed experiments for measuring longitudinal and transverse nuclear relaxation rates in highly paramagnetic systems will be also reviewed. Finally, we will address the use of extrinsic paramagnetic centers covalently bound to diamagnetic proteins, which contributed over the last twenty years to promote the applications of paramagnetic NMR well beyond the structural biology of metalloproteins.
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Clore, G. Marius. "Seeing the invisible by paramagnetic and diamagnetic NMR." Biochemical Society Transactions 41, no. 6 (November 20, 2013): 1343–54. http://dx.doi.org/10.1042/bst20130232.
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Sparsely populated transient states of proteins and their complexes play an important role in many biological processes including protein–protein and protein–DNA recognition, allostery, conformational selection, induced fit and self-assembly. These states are difficult to study as their low population and transient nature makes them effectively invisible to conventional structural and biophysical techniques. In the present article, I summarize recent NMR developments in our laboratory, including the use of paramagnetic relaxation enhancement, lifetime line broadening and dark-state exchange saturation transfer spectroscopy, that have permitted such sparsely populated states to be detected, characterized and, in some instances, visualized. I illustrate the application of these methods to the elucidation of mechanisms whereby transcription factors locate their specific target sites within an overwhelming sea of non-specific DNA, to the characterization of encounter complexes in protein–protein recognition, to large-scale interdomain motions involved in ligand binding, and to the interaction of monomeric amyloid β-peptide with the surface of amyloid protofibrils and the internal cavity surface of the chaperonin GroEL.
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Hunashal, Yamanappa, Cristina Cantarutti, Sofia Giorgetti, Loredana Marchese, Federico Fogolari, and Gennaro Esposito. "Insights into a Protein-Nanoparticle System by Paramagnetic Perturbation NMR Spectroscopy." Molecules 25, no. 21 (November 7, 2020): 5187. http://dx.doi.org/10.3390/molecules25215187.
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Background: The interaction between proteins and nanoparticles is a very relevant subject because of the potential applications in medicine and material science in general. Further interest derives from the amyloidogenic character of the considered protein, β2-microglobulin (β2m), which may be regarded as a paradigmatic system for possible therapeutic strategies. Previous evidence showed in fact that gold nanoparticles (AuNPs) are able to inhibit β2m fibril formation in vitro. Methods: NMR (Nuclear Magnetic Resonance) and ESR (Electron Spin Resonance) spectroscopy are employed to characterize the paramagnetic perturbation of the extrinsic nitroxide probe Tempol on β2m in the absence and presence of AuNPs to determine the surface accessibility properties and the occurrence of chemical or conformational exchange, based on measurements conducted under magnetization equilibrium and non-equilibrium conditions. Results: The nitroxide perturbation analysis successfully identifies the protein regions where protein-protein or protein-AuNPs interactions hinder accessibility or/and establish exchange contacts. These information give interesting clues to recognize the fibrillation interface of β2m and hypothesize a mechanism for AuNPs fibrillogenesis inhibition. Conclusions: The presented approach can be advantageously applied to the characterization of the interface in protein-protein and protein-nanoparticles interactions.
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Anthis, Nicholas J., and G. Marius Clore. "Visualizing transient dark states by NMR spectroscopy." Quarterly Reviews of Biophysics 48, no. 1 (January 20, 2015): 35–116. http://dx.doi.org/10.1017/s0033583514000122.
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AbstractMyriad biological processes proceed through states that defy characterization by conventional atomic-resolution structural biological methods. The invisibility of these ‘dark’ states can arise from their transient nature, low equilibrium population, large molecular weight, and/or heterogeneity. Although they are invisible, these dark states underlie a range of processes, acting as encounter complexes between proteins and as intermediates in protein folding and aggregation. New methods have made these states accessible to high-resolution analysis by nuclear magnetic resonance (NMR) spectroscopy, as long as the dark state is in dynamic equilibrium with an NMR-visible species. These methods – paramagnetic NMR, relaxation dispersion, saturation transfer, lifetime line broadening, and hydrogen exchange – allow the exploration of otherwise invisible states in exchange with a visible species over a range of timescales, each taking advantage of some unique property of the dark state to amplify its effect on a particular NMR observable. In this review, we introduce these methods and explore two specific techniques – paramagnetic relaxation enhancement and dark state exchange saturation transfer – in greater detail.
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Gong, Zhou, Shuai Yang, Qing-Fen Yang, Yue-Ling Zhu, Jing Jiang, and Chun Tang. "Refining RNA solution structures with the integrative use of label-free paramagnetic relaxation enhancement NMR." Biophysics Reports 5, no. 5-6 (November 15, 2019): 244–53. http://dx.doi.org/10.1007/s41048-019-00099-2.
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AbstractNMR structure calculation is inherently integrative, and can incorporate new experimental data as restraints. As RNAs have lower proton densities and are more conformational heterogenous than proteins, the refinement of RNA structures can benefit from additional types of restraints. Paramagnetic relaxation enhancement (PRE) provides distance information between a paramagnetic probe and protein or RNA nuclei. However, covalent conjugation of a paramagnetic probe is difficult for RNAs, thus limiting the use of PRE NMR for RNA structure characterization. Here, we show that the solvent PRE can be accurately measured for RNA labile imino protons, simply with the addition of an inert paramagnetic cosolute. Demonstrated on three RNAs that have increasingly complex topologies, we show that the incorporation of the solvent PRE restraints can significantly improve the precision and accuracy of RNA structures. Importantly, the solvent PRE data can be collected for RNAs without isotope enrichment. Thus, the solvent PRE method can work integratively with other biophysical techniques for better characterization of RNA structures.
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Larsen, Erik, Cristina Olivieri, Caitlin Walker, Manu V.S., Jiali Gao, David Bernlohr, Marco Tonelli, John Markley, and Gianluigi Veglia. "Probing Protein-Protein Interactions Using Asymmetric Labeling and Carbonyl-Carbon Selective Heteronuclear NMR Spectroscopy." Molecules 23, no. 8 (August 3, 2018): 1937. http://dx.doi.org/10.3390/molecules23081937.
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Protein-protein interactions (PPIs) regulate a plethora of cellular processes and NMR spectroscopy has been a leading technique for characterizing them at the atomic resolution. Technically, however, PPIs characterization has been challenging due to multiple samples required to characterize the hot spots at the protein interface. In this paper, we review our recently developed methods that greatly simplify PPI studies, which minimize the number of samples required to fully characterize residues involved in the protein-protein binding interface. This original strategy combines asymmetric labeling of two binding partners and the carbonyl-carbon label selective (CCLS) pulse sequence element implemented into the heteronuclear single quantum correlation (1H-15N HSQC) spectra. The CCLS scheme removes signals of the J-coupled 15N–13C resonances and records simultaneously two individual amide fingerprints for each binding partner. We show the application to the measurements of chemical shift correlations, residual dipolar couplings (RDCs), and paramagnetic relaxation enhancements (PRE). These experiments open an avenue for further modifications of existing experiments facilitating the NMR analysis of PPIs.
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Öster, Carl, Simone Kosol, Christoph Hartlmüller, Jonathan M. Lamley, Dinu Iuga, Andres Oss, Mai-Liis Org, et al. "Characterization of Protein–Protein Interfaces in Large Complexes by Solid-State NMR Solvent Paramagnetic Relaxation Enhancements." Journal of the American Chemical Society 139, no. 35 (August 25, 2017): 12165–74. http://dx.doi.org/10.1021/jacs.7b03875.
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Prestegard, J. H., H. M. Al-Hashimi, and J. R. Tolman. "NMR structures of biomolecules using field oriented media and residual dipolar couplings." Quarterly Reviews of Biophysics 33, no. 4 (November 2000): 371–424. http://dx.doi.org/10.1017/s0033583500003656.
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1. Introduction 3721.1 Residual dipolar couplings as a route to structure and dynamics 3721.2 A brief history of oriented phase high resolution NMR 3742. Theoretical treatment of dipolar interactions 3762.1 Anisotropic interactions as probes of macromolecular structure and dynamics 3762.1.1 The dipolar interaction 3762.1.2 Averaging in the solution state 3772.2 Ordering of a rigid body 3772.2.1 The Saupe order tensor 3782.2.2 Orientational probability distribution function 3802.2.3 The generalized degree of order 3802.3 Molecular structure and internal dynamics 3813. Inducing molecular order in high resolution NMR 3833.1 Tensorial interactions between the magnetic field and anisotropic magnetic susceptibilities 3833.2 Dilute liquid crystal media: a tunable source of order 3843.2.1 Bicelles : from membrane mimics to aligning media 3853.2.2 Filamentous phage 3873.2.3 Transfer of alignment from ordered media to macromolecules 3883.3 Magnetic field alignment 3893.3.1 Paramagnetic assisted alignment 3893.3.2 Advantages of using magnetic alignment 3894. The measurement of residual dipolar couplings 3914.1 Introduction 3914.2 Frequency based methods 3924.2.1 Coupling enhanced pulse schemes 3924.2.2 In phase anti-phase methods (IPAP): 1DNH couplings in proteins 3934.2.3 Exclusive correlated spectroscopy (E-COSY): 1DNH, 1DNC′ and 2DHNC′ 3954.2.4 Extraction of splitting values from the frequency domain 3964.3 Intensity based experiments 3974.3.1 J-Modulated experiments: the measurement of 1DCαHα in proteins 3974.3.2 Phase modulated methods 3994.3.3 Constant time COSY – the measurement of DHH couplings 3994.3.4 Systematic errors in intensity based experiments 4005. Interpretation of residual dipolar coupling data 4015.1 Structure determination protocols utilizing orientational constraints 4015.1.1 The simulated annealing approach 4015.1.2 Order matrix analysis of dipolar couplings 4025.1.3 A discussion of the two approaches 4025.2 Reducing orientational degeneracy 4035.2.1 Multiple alignment media in the simulated annealing approach 4045.2.2 Multiple alignment media in the order matrix approach 4055.3 Simplifying effects arising due to molecular symmetry 4065.4 Database approaches for determining protein structure 4076. Applications to the characterization of macromolecular systems 4086.1 Protein structure refinement 4086.2 Protein domain orientation 4096.3 Oligosaccharides 4136.4 Biomolecular complexes 4156.5 Exchanging systems 4167. Acknowledgements 4188. References 419Within its relatively short history, nuclear magnetic resonance (NMR) spectroscopy has managed to play an important role in the characterization of biomolecular structure. However, the methods on which most of this characterization has been based, Nuclear Overhauser Effect (NOE) measurements for short-range distance constraints and scalar couplings measurements for torsional constraints, have limitations (Wüthrich, 1986). For extended structures, such as DNA helices, for example, propagation of errors in the short distance constraints derived from NOEs leaves the relative orientation of remote parts of the structures poorly defined. Also, the low density of observable protons in contact regions of molecules held together by factors other than hydrophobic packing, leads to poorly defined structures. This is especially true in carbohydrate containing complexes where hydrogen bonds often mediate contacts, and in multi-domain proteins where the area involved in domain–domain contact can also be small. Moreover, most NMR based structural applications are concerned with the characterization of a single, rigid conformer for the final structure. This can leave out important mechanistic information that depends on dynamic aspects and, when motion is present, this can lead to incorrect structural representations. This review focuses on one approach to alleviating some of the existing limitations in NMR based structure determination: the use of constraints derived from the measurement of residual dipolar couplings (D).
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Möbius, Klaus, Wolfgang Lubitz, Nicholas Cox, and Anton Savitsky. "Biomolecular EPR Meets NMR at High Magnetic Fields." Magnetochemistry 4, no. 4 (November 6, 2018): 50. http://dx.doi.org/10.3390/magnetochemistry4040050.
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In this review on advanced biomolecular EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR regarding the measurement of interactions and dynamics of large molecules embedded in fluid-solution or solid-state environments. Our focus is on the characterization of protein structure, dynamics and interactions, using sophisticated EPR spectroscopy methods. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy to new horizons reaching millimeter and sub-millimeter wavelengths and 15 T Zeeman fields. Expanding traditional applications to paramagnetic systems, spin-labeling of biomolecules has become a mainstream multifrequency approach in EPR spectroscopy. In the high-frequency/high-field EPR region, sub-micromolar concentrations of nitroxide spin-labeled molecules are now sufficient to characterize reaction intermediates of complex biomolecular processes. This offers promising analytical applications in biochemistry and molecular biology where sample material is often difficult to prepare in sufficient concentration for NMR characterization. For multifrequency EPR experiments on frozen solutions typical sample volumes are of the order of 250 μL (S-band), 150 μL (X-band), 10 μL (Q-band) and 1 μL (W-band). These are orders of magnitude smaller than the sample volumes required for modern liquid- or solid-state NMR spectroscopy. An important additional advantage of EPR over NMR is the ability to detect and characterize even short-lived paramagnetic reaction intermediates (down to a lifetime of a few ns). Electron–nuclear and electron–electron double-resonance techniques such as electron–nuclear double resonance (ENDOR), ELDOR-detected NMR, PELDOR (DEER) further improve the spectroscopic selectivity for the various magnetic interactions and their evolution in the frequency and time domains. PELDOR techniques applied to frozen-solution samples of doubly spin-labeled proteins allow for molecular distance measurements ranging up to about 100 Å. For disordered frozen-solution samples high-field EPR spectroscopy allows greatly improved orientational selection of the molecules within the laboratory axes reference system by means of the anisotropic electron Zeeman interaction. Single-crystal resolution is approached at the canonical g-tensor orientations—even for molecules with very small g-anisotropies. Unique structural, functional, and dynamic information about molecular systems is thus revealed that can hardly be obtained by other analytical techniques. On the other hand, the limitation to systems with unpaired electrons means that EPR is less widely used than NMR. However, this limitation also means that EPR offers greater specificity, since ordinary chemical solvents and matrices do not give rise to EPR in contrast to NMR spectra. Thus, multifrequency EPR spectroscopy plays an important role in better understanding paramagnetic species such as organic and inorganic radicals, transition metal complexes as found in many catalysts or metalloenzymes, transient species such as light-generated spin-correlated radical pairs and triplets occurring in protein complexes of photosynthetic reaction centers, electron-transfer relays, etc. Special attention is drawn to high-field EPR experiments on photosynthetic reaction centers embedded in specific sugar matrices that enable organisms to survive extreme dryness and heat stress by adopting an anhydrobiotic state. After a more general overview on methods and applications of advanced multifrequency EPR spectroscopy, a few representative examples are reviewed to some detail in two Case Studies: (I) High-field ELDOR-detected NMR (EDNMR) as a general method for electron–nuclear hyperfine spectroscopy of nitroxide radical and transition metal containing systems; (II) High-field ENDOR and EDNMR studies of the Oxygen Evolving Complex (OEC) in Photosystem II, which performs water oxidation in photosynthesis, i.e., the light-driven splitting of water into its elemental constituents, which is one of the most important chemical reactions on Earth.
Dissertations / Theses on the topic "NMR, Paramagnetism, Protein Characterization"
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Caramia, Sara. "Purification and preliminary structural characterization by NMR spectroscopy of the "HoLaMa" DNA polymerase." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2017. http://amslaurea.unibo.it/14420/.
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HoLaMa is a Klenow sub-fragment lacking the 3’-5’ exonuclease domain, whose gene codes for residues 515-928 of Escherichia coli DNA polymerase I. The enzyme was designed in a previous study starting from different Klenow enzymes, with the aim of studying a mini-DNA polymerase with NMR spectroscopy. In the present work, we studied a new purification protocol for the production of HoLaMa in order to obtain an appropriate quantity for NMR analysis. We tested three different purification procedure and at the end, we collected 5.8 mg of HoLaMa (Volume 1.8 mL, concentration 67 μM). After the purification, we started the study of HoLaMa by NMR spectroscopy, focusing on the nature of enzyme-substrate interactions and studying the kinetics of the reaction. Our preliminary studies were designed to understand the characteristic NMR signals of HoLaMa under different conditions of temperature and buffer; finally, we also focused our analysis on the interactions between protein, DNA and nucleotides.
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Nishizawa, Mayu. "Physicochemical Characterization of Physiological Aspects of Protein Structure." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263680.
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京都大学新制・課程博士
博士(工学)
甲第23219号
工博第4863号
京都大学大学院工学研究科分子工学専攻
(主査)教授 田中 庸裕, 教授 近藤 輝幸, 准教授 菅瀬 謙治, 教授 佐藤 啓文
学位規則第4条第1項該当
Doctor of Philosophy (Engineering)
Kyoto University
DGAM
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MERLO, SILVIA. "Characterization of biomedical relevant ligand-protein interactions using Nuclear Magnetic Resonance (NMR) Spectroscopy." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2013. http://hdl.handle.net/10281/40953.
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NMR techniques allow to obtain structural information useful for the comprehension of biological processes. The aim of this work is to investigate interactions between biological macromolecules and binding partners, including other macromolecules, small ligands and therapeutically relevant compounds. These results will be exploited for the design and development of new potential drugs and medical devices.
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MACCHI, ELEONORA. "NMR as a tool for structural characterization of carbohydrates and glycan-protein interactions." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/69274.
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Il virus dell’influenza A è un virus a RNA formato da 8 geni, tre dei quali - emagglutinina (HA), neuramminidasi (NA) e polimerasi (PB) –, risultano particolarmente critici nell’infezione e nella trasmissione uomo-uomo. L’infezione inizia con il legame dell’HA del virus ai recettori glicanici presenti sulle cellule dell’ospite; questa interazione è altamente specifica, ed è governata dal tipo di legame tra l’acido sialico e il galattosio all’interno del recettore.
I recettori umani glicanici, siti di riconoscimento per i virus human-adapted, sono espressi principalmente nel tratto superiore dell’epitelio respiratorio umano e presentano un legame α2→6 tra l’acido sialico e il galattosio nell’estremità non riducente. I virus aviari invece, riconoscono recettori glicanici che presentano un legame α2→3 tra acido neuraminico e il galattosio.
Studi precedenti dell’interazione tra HA e trisaccaridi hanno dimostrato che sia la conformazione dei glicani, che il diverso tipo di legame tra acido sialico e galattosio sono fattori chiave per la regolazione dell’interazione. Partendo dalle sopracitate considerazioni questo lavoro di ricerca si è occupato di studiare la dinamica e la conformazione in soluzione di due pentasaccaridi, usati come modelli per il recettore aviario (LSTa, Neu5Ac-α(2→3)-Gal-β(1→3)-GlcNAc-β(1→3)-Gal-β(1→4)-Glc) e umano (LSTc, Neu5Ac-α(2→6)-Gal-β(1→4)-GlcNAc-β(1→3)-Gal-β(1→4)-Glc), utilizzando tecniche NMR (Nuclear Magnetic Resonance) e simulazioni di dinamica molecolare (MD). I nostri studi dimostrano che in soluzione i due recettori presentano diverse conformazioni, dinamiche e topologie. Queste peculiarità uniche comportano caratteristiche molecolari diverse per il riconoscimento di HA, dimostrando quindi la specificità dell’interazione tra recettore e emagglutinina.
La relazione tra la specificità dell’HA verso i recettori e la trasmissibilità del virus è stata precedentemente dimostrata usando il prototipo del virus SC18 (H1N1) A/South Carolina/1/1918. Combinando tecniche di Risonanza Magnetica Nucleare e di dinamica molecolare, abbiamo dimostrato come, durante l’interazione, il sito di binding dell’emagglutinina imponga differenti vincoli conformazionali al recettore. Il virus pandemico SC18, che presenta un’efficacia di trasmissione negli uomini molto alta, a confronto con il singolo (NY18, Asp225 → Gly) e doppio (AV18, Asp190 → Glu e Asp225 → Gly) mutante, impone maggiori vincoli alla conformazione del recettore umano, proprietà correlata all’affinità dell’interazione recettore-HA, misurata tramite saggi biochimici.
Questa relazione tra affinità e vincoli conformazionali imposti al recettore è stata osservata anche per il virus aviario-adattato AV18, il quale impone vincoli conformazionali maggiori al recettore aviario in confronto a quelli imposti a quest’ultimo da NY18. In particolare, è interessante osservare come emagglutinine differenti impongano vincoli conformazionali diversi a seconda che leghino recettori umani o aviari. In ultimo, abbiamo esteso il nostro studio a un virus meno pandemico, H7N9, e due suoi mutanti, i quali sono in grado di legare sia il recettore umano che aviario, allo scopo di capire come avviene l’interazione tramite l’utilizzo di tecniche NMR e di dinamica molecolare.
In questo studio descriviamo le basi strutturali dell’interazione tra l’emagglutinina di nuovi virus e i recettori umani e aviari, combinando l’approccio sperimentale a tecniche computazionali. Questa metodologia potrà essere usata come strumento utile per la sorveglianza di nuovi virus pandemici.
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Gustafsson, Robert. "Biophysical characterization of the *5 protein variant of human thiopurine methyltransferase by NMR spectroscopy." Thesis, Linköpings universitet, Molekylär Bioteknik, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-78526.
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Human thiopurine methyltransferase (TPMT) is an enzyme involved in the metabolism of thiopurine drugs, which are widely used in leukemia and inflammatory bowel diseases such as ulcerative colitis and Crohn´s disease. Due to genetic polymorphisms, approximately 30 protein variants are present in the population, some of which have significantly lowered activity. TPMT *5 (Leu49Ser) is one of the protein variants with almost no activity. The mutation is positioned in the hydrophobic core of the protein, close to the active site. Hydrogen exchange rates measured with NMR spectroscopy for N-terminally truncated constructs of TPMT *5 and TPMT *1 (wild type) show that local stability and hydrogen bonding patterns are changed by the mutation Leu49Ser. Most residues exhibit faster exchange rates and a lower local stability in TPMT *5 in comparison with TPMT *1. Changes occur close to the active site but also throughout the entire protein. Calculated overall stability is similar for the two constructs, so the measured changes are due to local stability. Protein dynamics measured with NMR relaxation experiments show that both TPMT *5 and TPMT *1 are monomeric in solution. Millisecond dynamics exist in TPMT *1 but not in TPMT *5, even though a few residues exhibit a faster dynamic. Dynamics on nanosecond to picosecond time scale have changed but no clear trends are observable.
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Hake, Michael James. "Spectroscopic Characterization of the Interaction of Nck Domains with the Epidermal Growth Factor Receptor Juxtamembrane Domain." Case Western Reserve University School of Graduate Studies / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1207340174.
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Silvennoinen, L. (Laura). "ERp57—Characterization of its domains and determination of solution structures of the catalytic domains." Doctoral thesis, University of Oulu, 2006. http://urn.fi/urn:isbn:9514280547.
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Abstract
The correct three dimensional structures of proteins are essential for their ability to function properly. Proteins start to fold as soon as they are synthesized in the ribosomes from activated amino acids. Many secreted, cell-surface, secretory pathway and endoplasmic reticulum (ER) lumenal proteins have in their amino acid sequence cysteine residues which form intra- and intermolecular disulfide bridges that stabilize the overall fold of the proteins and protein complexes. The formation of correct disulfide bonds is a complex process which takes place within the ER.
Protein disulfide isomerase (PDI) is the key enzyme in the formation and rearrangement of correct disulfide bonds in the ER. It is an archetypal and the best studied member of the PDI family, i.e. a group of ER proteins that resemble thioredoxin (TRX), a protein reductase, in their structure. PDI has a four domain a-b-b'-a' structure the a and a' domains having the catalytic activity and amino acid sequence similarity to TRX. In addition to its function as a thiol-disulfide oxidoreductase, PDI acts as the β subunit in two protein complexes: collagen prolyl 4-hydroxylase (C-P4H) and microsomal triglyceride transfer protein (MTP).
The closest homologue of PDI is the multifunctional enzyme and chaperone ERp57 that functions in concert with two lectins, calnexin (CNX) and calreticulin (CRT) specifically in the folding of proteins that have sugar moieties linked to them. ERp57 is 56% similar to PDI in its amino acid sequence and has also the four-domain architecture. Despite the high similarity in their structures ERp57 cannot substitute for PDI as the β subunit of C-P4H. The minimum requirement for the C-P4H tetramer assembly is fulfilled by domains b' and a' of PDI, while domains a and b enhance this function and can be substituted in part by those of ERp57.
Until very recently the structural information of any of the PDI family members, which contains the TRX active site was limited to solution structures of human PDI domains a and b. In this research the domain boundaries of the full length ERp57 were defined and the individual domains characterized. Furthermore the solution structures of the catalytically active domains a and a' of ERp57 were studied by nuclear magnetic resonance (NMR).
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Ahlner, Alexandra. "Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain." Doctoral thesis, Linköpings universitet, Kemi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-117076.
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Proteins are essential for all known forms of life and in many lethal diseases protein failure is the cause of the disease. To understand proteins and the processes they are involved in, it is valuable to know their structures as well as their dynamics and interactions. The structures may not be directly inspected because proteins are too small to be visible in a light microscope, which is why indirect methods such as nuclear magnetic resonance (NMR) spectroscopy have to be utilized. This method provides atomic information about the protein and, in contrast to other methods with similar resolution, the measurements are performed in solution resulting in more physiological conditions, enabling analysis of dynamics. Important dynamical processes are the ones on the millisecond timeframe, which may contribute to interactions of proteins and their catalysis of chemical reactions, both of significant value for the function of the proteins. To better understand proteins, not only do we need to study them, but also develop the methods we are using. This thesis presents four papers about improved NMR techniques as well as a fifth where the kinase domain of ephrinB receptor 2 (EphB2) has been studied regarding the importance of millisecond dynamics and interactions for the activation process. The first paper presents the software COMPASS, which combines statistics and the calculation power of a computer with the flexibility and experience of the user to facilitate and speed up the process of assigning NMR signals to the atoms in the protein. The computer program PINT has been developed for easier and faster evaluation of NMR experiments, such as those that evaluate protein dynamics. It is especially helpful for NMR signals that are difficult to distinguish, so called overlapped peaks, and the soft- ware also converts the detected signals to the indirectly measured physical quantities, such as relaxation rate constants, principal for dynamics. Next are two new versions of the Carr-Purcell-Maiboom-Gill (CPMG) dispersion pulse sequences, designed to measure millisecond dynamics in a way so that the signals are more separated than in standard experiments, to reduce problems with overlaps. To speed up the collection time of the data set, a subset is collected and the entire data set is then reconstructed, by multi-dimensional decomposition co-processing. Described in the thesis is also a way to produce suitably labeled proteins, to detect millisecond dynamics at Cα positions in proteins, using the CPMG dispersion relaxation experiment at lower protein concentrations. Lastly, the kinase domain of EphB2 is shown to be more dynamic on the millisecond time scale as well as more prone to interact with itself in the active form than in the inactive one. This is important for the receptor function of the protein, when and how it mediates signals. To conclude, this work has extended the possibilities to study protein dynamics by NMR spectroscopy and contributed to increased understanding of the activation process of EphB2 and its signaling mechanism.
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Ghosh, Madhumita. "Structural and biochemical characterization of proteins involved in cancer." [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=974284823.
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Pondaven, Simon Pierre. "Conformational Flexibility and Amyloid Core Characterization of Human Immunoglobulin Light Chain Domains by Multidimensional NMR Spectroscopy." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1354113457.
Full textBook chapters on the topic "NMR, Paramagnetism, Protein Characterization"
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Grondin, Julie M., David N. Langelaan, and Steven P. Smith. "Characterization of Protein-Carbohydrate Interactions by NMR Spectroscopy." In Methods in Molecular Biology, 143–56. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-6899-2_11.
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Hill, Justine M. "NMR Screening for Rapid Protein Characterization in Structural Proteomics." In Methods in Molecular Biology, 437–46. Totowa, NJ: Humana Press, 2008. http://dx.doi.org/10.1007/978-1-60327-058-8_29.
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Blackledge, Martin, Pau Bernadó, and Malene Ringkjøbing Jensen. "Atomic-Level Characterization of Disordered Protein Ensembles Using NMR Residual Dipolar Couplings." In Instrumental Analysis of Intrinsically Disordered Proteins, 89–106. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2010. http://dx.doi.org/10.1002/9780470602614.ch4.
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Goyal, Shaveta, Haina Qin, Liangzhong Lim, and Jianxing Song. "Insoluble Protein Characterization by Circular Dichroism (CD) Spectroscopy and Nuclear Magnetic Resonance (NMR)." In Methods in Molecular Biology, 371–85. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-2205-5_21.
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McCullough, Christopher, Phani Kumar Pullela, Sang-Choul Im, Lucy Waskell, and Daniel Sem. "The Synthesis, Characterization, and Application of 13C-Methyl Isocyanide as an NMR Probe of Heme Protein Active Sites." In Methods in Molecular Biology, 51–59. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-321-3_4.
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Satterlee, James D., Christine M. Suquet, Marina I. Savenkova, and Chenyang Lian. "Proton NMR Characterization of Recombinant Ferric Heme Domains of the Oxygen Sensors FixL and Dos: Evidence for Protein Heterogeneity." In ACS Symposium Series, 244–57. Washington, DC: American Chemical Society, 2003. http://dx.doi.org/10.1021/bk-2003-0858.ch013.
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Sljoka, Adnan. "Structural and Functional Analysis of Proteins Using Rigidity Theory." In Sublinear Computation Paradigm, 337–67. Singapore: Springer Singapore, 2021. http://dx.doi.org/10.1007/978-981-16-4095-7_14.
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AbstractOver the past two decades, we have witnessed an unprecedented explosion in available biological data. In the age of big data, large biological datasets have created an urgent need for the development of bioinformatics methods and innovative fast algorithms. Bioinformatics tools can enable data-driven hypothesis and interpretation of complex biological data that can advance biological and medicinal knowledge discovery. Advances in structural biology and computational modelling have led to the characterization of atomistic structures of many biomolecular components of cells. Proteins in particular are the most fundamental biomolecules and the key constituent elements of all living organisms, as they are necessary for cellular functions. Proteins play crucial roles in immunity, catalysis, metabolism and the majority of biological processes, and hence there is significant interest to understand how these macromolecules carry out their complex functions. The mechanical heterogeneity of protein structures and a delicate mix of rigidity and flexibility, which dictates their dynamic nature, is linked to their highly diverse biological functions. Mathematical rigidity theory and related algorithms have opened up many exciting opportunities to accurately analyse protein dynamics and probe various biological enigmas at a molecular level. Importantly, rigidity theoretical algorithms and methods run in almost linear time complexity, which makes it suitable for high-throughput and big-data style analysis. In this chapter, we discuss the importance of protein flexibility and dynamics and review concepts in mathematical rigidity theory for analysing stability and the dynamics of protein structures. We then review some recent breakthrough studies, where we designed rigidity theory methods to understand complex biological events, such as allosteric communication, large-scale analysis of immune system antibody proteins, the highly complex dynamics of intrinsically disordered proteins and the validation of Nuclear Magnetic Resonance (NMR) solved protein structures.
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Kamran, Fozia, Junus Salampessy, and Narsimha Reddy. "Application of NMR Spectroscopy for Structural Characterization of Bioactive Peptides Derived from Food Protein." In Applications of NMR Spectroscopy, 3–76. BENTHAM SCIENCE PUBLISHERS, 2016. http://dx.doi.org/10.2174/9781681082875116050003.
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Wemmer, D. "Design and Characterization of New Sequence Specific DNA Ligands." In Biological NMR Spectroscopy. Oxford University Press, 1997. http://dx.doi.org/10.1093/oso/9780195094688.003.0026.
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During the early 1980s there were two developments which lead to our studies of sequence specific DNA ligands. The first was the development of sequential assignment methods based on 2D NMR spectra which allowed complete assignment of resonances for proteins (Wüthrich, 1986). The assignments in turn allowed determination of many structural restraints through interpretation of NOESY crosspeaks and coupling constants from COSY type spectra. The second advance was the improvement of the chemistry for direct synthesis of DNA oligomers. With multimilligram samples of DNA oligomers available sequential assignment methods for DNA, paralleling those for proteins, were also worked out. Again with assignments came the possibility of determining DNA structures in solution. Howeverfor double stranded, Watson-Crick paired DNAs the structure can be reasonably approximated by the standard B-form model derived from fiber diffraction. The accurate determination of local conformational features has been somewhat difficult using NMR since tertiary contacts (as are so valuable in determining protein structures) do not occur. However with careful quantitative analysis some of the local details of structure can be determined. These NMR methods also offered the possibility of trying to understand the structural basis for binding of ligands to DNA oligomers. In order to make welldefined complexes we wanted to start with a compound that showed some sequence specificity in binding, and selected distamycin (shown below), a polypyrrole antibiotic which was known to have preference for binding to A-T rich DNA sequences. A close relative, netropsin, had been studied by Dinshaw Patel who showed that the binding is in the minor groove by identifying an NOE between a proton of the ligand and an adenosine H2 in the center of the minor groove (Patel, 1982). We began by making a complex with the self-complementary DNA oligomer: 5'-CGCGAATTCGCG-3', which had been studied extensively by X-- ray crystallography, and also by NMR. Distamycin did form a well-defined complex with this DNA, which was is slow exchange with free DNA during titrations (Klevit et al., 1986).
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Riek, Roland, Simone Hornemann, Gerhard Wider, Rudi Glockshuber, and Kurt Wüthrich. "NMR characterization of the full-length recombinant murine prion protein, mPrP(23–231)." In NMR with Biological Macromolecules in Solution, 120–26. WORLD SCIENTIFIC, 2021. http://dx.doi.org/10.1142/9789811235795_0015.
Full textReports on the topic "NMR, Paramagnetism, Protein Characterization"
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Shomer, Ilan, Ruth E. Stark, Victor Gaba, and James D. Batteas. Understanding the hardening syndrome of potato (Solanum tuberosum L.) tuber tissue to eliminate textural defects in fresh and fresh-peeled/cut products. United States Department of Agriculture, November 2002. http://dx.doi.org/10.32747/2002.7587238.bard.
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The project sought to understand factors and mechanisms involved in the hardening of potato tubers. This syndrome inhibits heat softening due to intercellular adhesion (ICA) strengthening, compromising the marketing of industrially processed potatoes, particularly fresh peeled-cut or frozen tubers. However, ICA strengthening occurs under conditions which are inconsistent with the current ideas that relate it to Ca-pectate following pectin methyl esterase (PME) activity or to formation of rhamnogalacturonan (RG)-II-borate. First, it was necessary to induce strengthening of the middle lamellar complex (MLX) and the ICA as a stress response in some plant parenchyma. As normally this syndrome does not occur uniformly enough to study it, we devised an efficient model in which ICA-strengthening is induced consistently under simulated stress by short-chain, linear, mono-carboxylic acid molecules (OAM), at 65 oC [appendix 1 (Shomer&Kaaber, 2006)]. This rapid strengthening was insufficient for allowing the involved agents assembly to be identifiable; but it enabled us to develop an efficient in vitro system on potato tuber parenchyma slices at 25 ºC for 7 days, whereas unified stress was reliably simulated by OAMs in all the tissue cells. Such consistent ICA-strengthening in vitro was found to be induced according to the unique physicochemical features of each OAM as related to its lipophilicity (Ko/w), pKa, protonated proportion, and carbon chain length by the following parameters: OAM dissociation constant (Kdiss), adsorption affinity constant (KA), number of adsorbed OAMs required for ICA response (cooperativity factor) and the water-induced ICA (ICAwater). Notably, ICA-strengthening is accompanied by cell sap leakage, reflecting cell membrane rupture. In vitro, stress simulation by OAMs at pH<pKa facilitated the consistent assembly of ICAstrengthening agents, which we were able to characterize for the first time at the molecular level within purified insoluble cell wall of ICA-strengthened tissue. (a) With solid-state NMR, we established the chemical structure and covalent binding to cell walls of suberin-like agents associated exclusively with ICA strengthening [appendix 3 (Yu et al., 2006)]; (b) Using proteomics, 8 isoforms of cell wall-bound patatin (a soluble vacuolar 42-kDa protein) were identified exclusively in ICA-strengthened tissue; (c) With light/electron microscopy, ultrastructural characterization, histochemistry and immunolabeling, we co-localized patatin and pectin in the primary cell wall and prominently in the MLX; (d) determination of cell wall composition (pectin, neutral sugars, Ca-pectate) yielded similar results in both controls and ICA-strengthened tissue, implicating factors other than PME activity, Ca2+ or borate ions; (e) X-ray powder diffraction experiments revealed that the cellulose crystallinity in the cell wall is masked by pectin and neutral sugars (mainly galactan), whereas heat or enzymatic pectin degradation exposed the crystalline cellulose structure. Thus, we found that exclusively in ICA-strengthened tissue, heat-resistant pectin is evident in the presence of patatin and suberinlike agents, where the cellulose crystallinity was more hidden than in fresh control tissue. Conclusions: Stress response ICA-strengthening is simulated consistently by OAMs at pH< pKa, although PME and formation of Ca-pectate and RG-II-borate are inhibited. By contrast, at pH>pKa and particularly at pH 7, ICA-strengthening is mostly inhibited, although PME activity and formation of Ca-pectate or RG-II-borate are known to be facilitated. We found that upon stress, vacuolar patatin is released with cell sap leakage, allowing the patatin to associate with the pectin in both the primary cell wall and the MLX. The stress response also includes formation of covalently bound suberin-like polyesters within the insoluble cell wall. The experiments validated the hypotheses, thus led to a novel picture of the structural and molecular alterations responsible for the textural behavior of potato tuber. These findings represent a breakthrough towards understanding of the hardening syndrome, laying the groundwork for potato-handling strategies that assure textural quality of industrially processed particularly in fresh peeled cut tubers, ready-to-prepare and frozen preserved products.