1,720,976 research outputs found
Molecular Modeling of the Interaction of Protein L with Antibodies
Full text linkProtein L (PpL) is a bacterial protein which is used in the affinity chromatography stage of the production of monoclonal antibodies because of its ability to form high affinity complexes with the light chains of immunoglobulins. In the present work, the binding interfaces between one domain of PpL and antigen-binding fragments (Fab) have been investigated adopting molecular dynamics with the aim of determining the binding contribution of the residues located at the Fab-PpL interface. Because it is known that PpL binds antibodies through two distinct binding sites with different affinities, simulations were performed for both sites to determine interaction free energies to assess the relative binding contribution of the two sites. Mutational studies were then performed only on the dominant binding site. The binding free energy was evaluated with the molecular mechanics Poisson-Boltzmann surface area (MMPBSA) and umbrella sampling/weighted histogram analysis methods. Key residues for the formation of the dominant binding site complex were identified by means of alanine scanning performed both for the Fab and PpL domains. Residues of the light chain of the antibody that contribute most to binding were found to be located between SER7 and VAL13. Four residues from PpL are important for the stability of the complex: PHE839, LYS840, GLU849, and TYR853. Three residues of PpL that do not contribute to the interaction were mutated to histidine (HIS), which changes its protonation state as a function of pH, to find whether this could allow us to control the binding interaction energy. This can be useful in the elution stage of the affinity chromatography purification of antibodies if PpL is used as a ligand. These residues are GLN835, THR836, and ALA837. Molecular dynamics simulations with both protonated and unprotonated HIS were performed to mimic how changing pH may reflect on protein- ligand interaction energies. The MMPBSA approach was used to evaluate the variation of the affinity of the mutated systems with reference to the wild type. Our results show that these mutations could help in disrupting the complex under acidic conditions without impairing the affinity of PpL for the light chains at higher pHs
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Development of methodologies for an accurate and efficient estimation of the interaction free energy in protein-protein and protein-ligand systems
No full textIn questo lavoro di tesi tre diversi sistemi sono stati investigate con lo scopo di stimare l’energia libera di legame. Per ogni sistema, metodi disponibili in letteratura sono stati utilizzati per ottenere una stima efficiente ed accurata dell’energia di interazione.
Il primo sistema è un complesso proteina-ligando, formato da un frammento Fab di un’immunoglobulina e una proteina commercialmente utilizzata nella fase di purificazione degli anticorpi monoclonali con cromatografia d’affinità. In questo lavoro i complessi tra un dominio della proteina L e i frammenti Fab sono stati studiati investigati usando simulazioni di dinamica molecolare per valutare il contributo all’affinità di legame dei residui della proteina L e dei frammenti Fab. L’effetto di un piccolo numero di mutazioni a istidina e del loro stato di protonazione sull’affinità è stato investigato per individuare mutazioni del ligando cha possano permettere l’uso di condizioni di eluzioni meno aspre durante la purificazione, in quanto il pH basso, tra 2 e 3.5, utilizzato può indurre aggregazione e cambi conformazionali negli anticorpi.
Il secondo caso studio è l’interazione tra due proteine cariche, Lisozima e Chimitripsinogeno A, e una superficie carica. Qui, due metodologie per la stima delle energie libere di interazione con diversi libelli di accuratezza e costo computazionale sono stati combinati. Il risultato è un protocollo che permette una stima rapida e accurata dell’energia di legame tra le proteine e la superficie. I sistemi investigati sono modelli di una fase di purificazione per cromatografia a scambio ionico delle due proteine di interesse. I valori calcolati sono stati poi utilizzati per stimare i fattori di ritenzione del processo cromatografico per avere un confronto tra i risultati teorici e i valori sperimentali.
L’ultimo sistema investigato è la formazione di aggregati tra due catene di poliglutammine. Questi particolari polipeptidi sono causa di malattie neurodegenerative, come la malattia di Huntington, e molte forme di atassia spinocerebellare. Il meccanismo per cui le catene di poliglutammine portano ai sintomi tipici di queste malattie non è ancora stato compreso a pieno. È noto che catene di polyQ con una lunghezza patologica formano aggregati contenenti strutture a beta foglietto simili alle fibrille amiloidi, ma la loro esatta struttura non è ancora nota, a causa della difficoltà di ottenere dati ad alta risoluzione degli aggregati. In questo lavoro proponiamo un modello per la formazione degli aggregati, usando un metodo di enhanced sampling, che permette di ottenere informazioni su un possibile meccanismo di formazione degli aggregati, e che offre la possibilità di stimare l’energia libera di formazione del complesso.In this thesis three different systems have been investigated with the aim of evaluating the binding free energy. For each system methods available in literature have been used to efficiently and accurately study the interaction.
The first system is a protein-ligand interaction, formed by a Fab fragment of immunoglobulin and a protein commercially used in the affinity chromatography step of the purification of monoclonal antibodies. In this work the complexes between one domain of protein L and Fab fragments have been investigated using molecular dynamics simulations to evaluate the contributions to the binding affinity of the residues of both protein L and Fab fragments. The effect of a small number of single point mutations to histidine and their protonation state on the affinity has been investigated to find mutations of the affinity ligand that could allow the use of milder conditions during the elution process of an affinity chromatography purification cycle, since the low pH used to recover the product, between 2 and about 3.5, can induce aggregation and conformational changes in the antibodies.
The second case study is the interaction between two charged proteins, Lysozyme and Chymotrypsinogen A, and a charged surface. Here two methodologies for the estimation of the binding free energy of interaction with different levels of accuracy and different computational costs, have been combined. The result is a protocol that allows a fast and accurate estimation of the binding free energy between the proteins and the surface. The systems investigated are models of a IEX chromatography phase for the purification of the two proteins of interest. Calculated values have been used to estimate the retention factor of the chromatography process in order to compare the theoretical results with the experimental values.
The last system investigated is the formation of aggregates between two polyglutamine chains. These peculiar polypeptides are cause of a number of neurodegenerative diseases, like Huntington’s disease, Kennedy’s disease, dentatorubral-pallidoluysian atrophy, and many forms of spinocerebellar ataxia. The mechanism by which polyglutamine chains lead to the symptoms typical of these diseases is not yet fully understood. It is known that pathological polyQ chains form aggregates containing β-sheets structure in a amyloid-like fashion, but their structure is still not know, due to the difficulty of obtaining high resolution data of the aggregates. In this work we propose a model of the formation of aggregates, using an enhanced sampling technique, that allows to gain insight in a possible mechanism of formation of the aggregate, and offer the possibility of estimating the binding free energy for the formation of the complex.DIPARTIMENTO DI CHIMICA, MATERIALI E INGEGNERIA CHIMICA "GIULIO NATTA"29MANCA, DAVIDEFRASSOLDATI, ALESSI
Modellazione molecolare dell'interazione tra proteina L ed il dominio variabile degli anticorpi
No full textLAUREA MAGISTRALEGli anticorpi monoclonali sono tra i più importanti prodotti farmaceutici in termini di potenziale terapeutico e di mercato e il loro costo è dovuto per il 50-80% alla fase di purificazione, di cui uno dei passaggi è la cromatografia di affinità. In questo lavoro di tesi viene proposto un nuovo modello dell'interazione tra la proteina L del Peptostreptococcus magnus, usata nella purificazione degli anticorpi, e le catene variabili delle immunoglobuline. Il modello è stato convalidato confrontandolo con i dati sperimentali disponibili in letteratura. E' stata poi eseguita una scansione di alanine per valutare gli amminoacidi che contribuiscono maggiormente all'affinità di legame, anche in questo caso i risultati ottenuti sono stati confrontati con i dati disponibili in letteratura. Infine sono state proposte tre mutazioni della proteina L in cui un amminoacido è stato mutato ad istidina, con lo scopo di migliorare l'affinità di interazione e facilitare la fase di eluizione durante la fase di purificazione per cromatografia di affinità. Queste predizioni sono suscettibili di prova sperimentale
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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