Cellular life, as we know it, is absolutely dependent on biological membranes; remarkable superstructures made of lipids and proteins. For example, all living cells are surrounded by at least one membrane that protects the cell and holds it together. The proteins that are embedded in the membranes carry out a wide variety of key functions, from nutrient uptake and waste disposal to cellular respiration and communication. In order to function accurately, any integral membrane protein needs to be inserted into the cellular membrane where it belongs, and in that particular membrane it has to attain its proper structure and find partners that might be required for proper function. All membrane proteins have evolved to be inserted in a specific overall orientation, so that e.g. substrate-binding parts are exhibited on the ‘right side’ of the membrane. So, what determines in which way a membrane protein is inserted? Are all membrane proteins inserted just so? The focus of this thesis is on these fundamental questions: how, and when, is the overall orientation of a membrane protein established? A closer look at the inner membrane proteome of the familiar gram-negative bacterium Escherichia coli revealed a small group of proteins that, oddly enough, seemed to be able to insert into the membrane in two opposite orientations. We could show that these dual-topology membrane proteins are delicately balanced, and that even the slightest manipulations make them adopt a fixed orientation in the membrane. Further, we show that these proteins are topologically malleable until the very last residue has been synthesized, implying interesting questions about the topogenesis of membrane proteins in general. In addition, by looking at the distribution of homologous proteins in other organisms, we got some ideas about how membrane proteins might evolve in size and complexity. Structural data has revealed that many membrane bound transporters have internal, inverted symmetries, and we propose that perhaps some of these proteins derive from dual-topology ancestors. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Bcl-XL is a membrane-associated protein that inhibits programmed cell death (apoptosis) in mammalian cells. Very little is known about the membrane topology of Bel-XL or how its association with membranes contributes to its function. It was the aim of this thesis to use fluorescence spectroscopy to investigate the location of a specific amino acid ofBcl-XL relative to the membrane. Bel-XL purified from E. coli could bind both to large unilamellar vesicles and endoplasmic reticulum (ER) microsomes isolated from canine pancreas. A cysteine residue at position 151 in Bcl-XL could be covalently labelled with the environmentally sensitive fluorescent molecule NBD. Emission intensity measurements in the presence and absence of membranes, combined with aqueous and lipophilic quenching experiments, indicate that Cys 151 is inserted into the interior of the membrane bilayer when Bcl-XL is bound to membranes. The methods outlined in this thesis form the basis for an experimental system that can be used to determine the membrane topology ofBclXL under a variety of conditions. / Thesis / Master of Science (MSc)

No description available.

Advances in protein topology mapping methods are urgently needed to complement the wealth of interactome data that is presently being generated at a rapid pace. Chemical crosslinking followed by mass spectrometry (MS) has evolved over the last decade as an attractive method for protein topology and interface mapping, and holds great promise as a counterpart to modern interactome studies in the field of proteomics. Furthermore, stabilization of proteins and protein complexes with crosslinking offers many advantages over high-resolution structural mapping methods, including the ability to study protein topologies in vivo. The reliance on direct detection of crosslinked peptides, however, continues to pose challenges to protein topology and interface mapping with chemical crosslinking plus MS. The present body of work aimed to develop a novel generic methodology that utilizes chemical crosslinking, cyanogen bromide (CNBr) cleavage and MS for the low-resolution mapping of protein topologies and interfaces. Through such low-resolution mapping of crosslinked regions, this novel strategy overcomes limitations associated with the direct detection of crosslinked peptides. Following optimization of various steps, the present method was validated with the bacterial DNA-directed RNA polymerase core complex and was subsequently applied to probe the tetrameric assembly of yeast Skp1p-Cdc4p heterodimers. Further improvements were made through the enrichment of crosslinked CNBr-cleaved protein fragments prior to their identification via MS. Two enrichment strategies were developed which depended upon the conjugation of tags to CNBr-cleaved peptide C-termini followed by either tandem affinity purification or tandem reversed-phase HPLC purification. These strategies were successfully applied for the efficient purification of disulfide-linked peptides from peptide mixtures. It is expected that the potential to achieve sensitive mapping of topologies and interfaces of multi-subunit protein complexes in vivo, in combination with further enhancements to permit studies on complex protein samples, will extend the utility of this method to complement large-scale interactome studies.

Advances in protein topology mapping methods are urgently needed to complement the wealth of interactome data that is presently being generated at a rapid pace. Chemical crosslinking followed by mass spectrometry (MS) has evolved over the last decade as an attractive method for protein topology and interface mapping, and holds great promise as a counterpart to modern interactome studies in the field of proteomics. Furthermore, stabilization of proteins and protein complexes with crosslinking offers many advantages over high-resolution structural mapping methods, including the ability to study protein topologies in vivo. The reliance on direct detection of crosslinked peptides, however, continues to pose challenges to protein topology and interface mapping with chemical crosslinking plus MS. The present body of work aimed to develop a novel generic methodology that utilizes chemical crosslinking, cyanogen bromide (CNBr) cleavage and MS for the low-resolution mapping of protein topologies and interfaces. Through such low-resolution mapping of crosslinked regions, this novel strategy overcomes limitations associated with the direct detection of crosslinked peptides. Following optimization of various steps, the present method was validated with the bacterial DNA-directed RNA polymerase core complex and was subsequently applied to probe the tetrameric assembly of yeast Skp1p-Cdc4p heterodimers. Further improvements were made through the enrichment of crosslinked CNBr-cleaved protein fragments prior to their identification via MS. Two enrichment strategies were developed which depended upon the conjugation of tags to CNBr-cleaved peptide C-termini followed by either tandem affinity purification or tandem reversed-phase HPLC purification. These strategies were successfully applied for the efficient purification of disulfide-linked peptides from peptide mixtures. It is expected that the potential to achieve sensitive mapping of topologies and interfaces of multi-subunit protein complexes in vivo, in combination with further enhancements to permit studies on complex protein samples, will extend the utility of this method to complement large-scale interactome studies.

>Magister Scientiae - MSc / Acute respiratory infections (ARI) continue to be the leading cause of acute illnesses worldwide and remain the most important cause of infant and young children mortality. Many viruses such as rhinoviruses, influenza viruses, parainfluenza viruses, respiratory syncytial viruses, adenoviruses and coronaviruses are deemed to be the etiological agents responsible for ARI’s in children. The recently discovered coronaviruses HCoV-HKU1 and HCoV-NL63 contribute significantly to the hospitalization of children with ARI’s. HCoV-NL63 was first identified in 2004, as the pathogen responsible for the hospitalization of a 7 month old child presenting with coryza, conjunctivitis and fever. Since then a significant amount of knowledge has been gained in the clinical spectrum on this virus, however HCoV-NL63 is still not well characterized on the molecular and proteomic level. This dissertation focuses on bringing about this characterization by cloning the HCoV-NL63 Nucleocapsid gene to be expressed in a bacterial system and transfecting the Nucleocapsid, Membrane and Envelope genes into a Mammalian cell culture system in order for its respective proteins to be expressed. With the use of Bioinformatic analytic tools certain characteristics of HCoV-NL63 Nucleocapsid, Membrane and Envelope proteins are able to be identified, as well as certain motifs and/or regions that are important in the functioning of these proteins. By comparing the results obtained for HCoV-NL63 N,M and E to other well studied coronavirus homologous will enlighten us on the potential role(s) of these proteins in determining HCoV-NL63 pathogenicity and infectivity. vi Although certain functions of these proteins can be deduced by the means of bioinformatics analysis, it is still imperative for it to be extensively characterized In Vitro. This will therefore form a fundamental step in the development of many other projects, which unfortunately fall outside the scope of this M.Sc thesis.

6 Abstract This master's thesis provides study of individual helixes from C-terminal pore-forming domain (CTD) of colicin U and their behavior in lipid bilayer on atomic level. For this purpose the all-atom molecular simulation method was used. Later the study was extended an applied on CTD of published structures of other pore-forming colicins. On the base of study extension the ability of disruption of lipid bilayer integrity by helixes H1 and H10 was successfully observed. Helix H1 was synthesized and its activity was experimentally proved on black lipid membranes. The other helixes are often too short to be able to keep position in lipid bilayer and their behavior could be affected by artificial termini, therefore they were not synthesized. The MD simulations of pairs of helixes show that structure stability and their ability to stay in the membrane depends on binding partners. The results of the thesis show the importance of H10 for colicin pore-formation, which has not been observed yet. The results also support the toroidal pore model suggested previously for colicin E1. The results prove that colicins contain specific secondary structures, which are able to disrupt the inner bacterial membrane not only in its native form but also when artificially separated from the rest of the protein. Klíčová...

Para que el proceso infeccioso de un virus de plantas tenga éxito la progenie viral tiene que propagarse desde las primeras células infectadas al resto de la planta; inicialmente se moverá célula a célula a través de los plasmodesmos (PDs) hasta alcanzar el sistema vascular, lo cual le permitirá invadir las partes distales de la planta. En este proceso, las proteínas de movimiento (MPs), junto con la colaboración de otros actores secundarios, desempeñan un papel relevante. El conocimiento de la posible asociación de las MPs con estructuras u orgánulos celulares así como de la interacción con factores del huésped es de vital importancia para poder desarrollar estrategias antivirales que permitan una mejora en la producción de los cultivos. Además, este tipo de estudios no sólo han posibilitado un mayor conocimiento de las respuestas al estrés en plantas sino que han sido pioneros en desentrañar los mecanismos de translocación intercelular de factores celulares implicados en los procesos de desarrollo de las plantas. Las MPs virales se clasifican en familias/grupos en función de su grado de similitud. Los virus, cuyas MPs pertenecen a la Superfamilia 30K, expresan una única MP encargada de orquestar el movimiento intra- e intercelular de genoma viral. En el Capítulo 1 de la presente Tesis se ha caracterizado la asociación de la MP del Virus del mosaico del tabaco (TMV), miembro tipo de la familia 30K, al sistema de endomembranas. Mediante el uso de aproximaciones in vivo se ha estudiado la eficiencia de inserción de sus regiones hidrofóbicas (HRs) en la membrana del retículo endoplasmático (ER). Nuestros resultados demuestran que ninguna de las dos HRs de la MP es capaz de atravesar las membranas biológicas y que la alteración de la hidrofobicidad de la primera HR es suficiente para modificar su asociación a la membrana. En base a los resultados obtenidos, proponemos un modelo topológico en el cual la MP del TMV se encontraría fuertemente asociada a la cara citosólica de la membrana del ER, sin llegar a atravesarla. La observación de que i), el modelo propuesto es compatible con otros motivos, previamente caracterizados, de la MP de TMV y ii), concuerda con la topología descrita para otras MPs de la familia 30K, permite cuestionar el modelo establecido desde el año 2000 para la MP de TMV así como predecir, en base a la conservada estructura secundaria de las MPs de esta familia, una topología similar para todos sus componentes. Para el transporte intercelular de los virus de plantas se han descrito tres modelos en base a la capacidad de transportar complejos ribonucloeprotéicos, a través de PD modificados, formados por el RNA viral y la MP (ej. MP de TMV) más la proteína de cubierta (ej. MP del virus del mosaico del pepino, CMV) o la capacidad de transportar viriones a través estructuras tubulares formadas por la MP (ej. MP del Virus del mosaico del caupí, CPMV). A pesar de las diferencias observadas entre los tres modelos, las MPs representativas de cada uno de ellos pertenecen a la misma familia 30K y son funcionalmente intercambiables (MPs de TMV, CMV, CPMV, Virus del mosaico del Bromo -BMV- o Virus de los anillos necróticos de los prunus -PNRSV-) por la MP del Virus del mosaico de la alfalfa (AMV), para el transporte a corta distancia. Con el objeto de comprender la versatilidad que presentan las MPs en cuanto al movimiento viral, hemos analizado la capacidad de estas MPs heterólogas de transportar sistémicamente el genoma quimérico del AMV. El estudio ha revelado que todas las MPs analizadas permiten el transporte del genoma quimera a las partes distales de la planta, independientemente del modelo descrito para el transporte a corta distancia, aunque requieren la extensión de los 44 aminoácidos C-terminales de la MP del AMV. Además, para todas las ellas, excepto para la MP del TMV, se ha establecido una relación entre la capacidad de movimiento local y la presencia del virus en las hojas no inoculadas de la planta, indicando la existencia de un umbral de transporte célula a célula, por debajo del cual, el virus es incapaz de invadir sistémicamente la planta. Durante el proceso de infección viral, las MPs interaccionan tanto con otras proteínas de origen viral como de la planta huésped. La interacción entre las MPs y dichos factores de la planta afectan a la patogénesis viral, facilitando u obstaculizando el movimiento intra- o intercelular del virus. En el Capítulo 3 del presente trabajo hemos demostrado la interacción entre la MP del AMV y dos miembros de la familia de Patellinas de arabidopsis, Patellin 3 (atPATL3) y Patellin 6 (atPATL6), mediante el sistema de los dos híbridos de levadura y ensayos de reconstitución bimolecular de la fluorescencia. Nuestros resultados, en general, demuestran que la interacción entre la MP-PATLs obstaculizaría un correcto direccionamiento de la MP al PD, dando lugar a un movimiento intracelular menos eficiente de los complejos virales, que forma la MP, y disminuyendo el movimiento célula a célula del virus. Podríamos estar hablando de un posible mecanismo de defensa de la planta, dirigido a evitar la invasión sistémica del huésped. En este sentido, las MPs virales pueden ser buenos candidatos para el desarrollo de estrategias antivirales dado que cualquier respuesta de defensa de la planta que, a priori, reduzca el transporte célula a célula del virus, puede representar la diferencia entre una infección local o sistémica, como hemos observado en el Capítulo 2 del presente trabajo. Los virus, a su vez, también son capaces de evolucionar hacia variantes más eficaces, que permitan superar las diferentes barreras defensivas de la planta huésped. En este contexto hemos identificado a la MP del Virus del bronceado del tomate (TSWV) como determinante de avirulencia en la resistencia mediada por el gen Sw-5. Del mismo modo, comprobamos que el cambio de 1-2 residuos de amino ácidos de la MP de TSWV fue suficiente para superar la resistencia pero que a la vez, y posiblemente debido a las altas restricciones que conlleva el reducido genoma de un virus, afectaron a la eficiencia de la MP. / Peiró Morell, A. (2014). Proteínas de movimiento de la familia 30K:interacción con membranas biológicas y factores proteicos y su implicación en el transporte viral [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48471 / TESIS