Structural study of the membrane protein MscL using cell-free expression and solid-state

a b s t r a c t High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75 kDa pentameric a-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR

Labelling strategy and membrane characterization of marine bacteria Vibrio splendidus by in vivo 2H NMR

Vibrio splendidus is a marine bacterium often considered as a threat in aquaculture hatcheries where it is responsible for mass mortality events, notably of bivalves' larvae. This bacterium is highly adapted to dynamic salty ecosystems where it has become an opportunistic and resistant species. To characterize their membranes as a first and necessary step toward studying bacterial interactions with diverse molecules, we established a labelling protocol for in vivo 2H solid-state nuclear magnetic resonance (SS-NMR) analysis of V. splendidus. 2H SS-NMR is a useful tool to study the organization and dynamics of phospholipids at the molecular level, and its application to intact bacteria is further advantageous as it allows probing acyl chains in their natural environment and study membrane interactions. In this study, we showed that V. splendidus can be labelled using deuterated palmitic acid, and demonstrated the importance of surfactant choice in the labelling protocol. Moreover, we assessed the impact of lipid deuteration on the general fitness of the bacteria, as well as the saturated-to-unsaturated fatty acid chains ratio and its impact on the membrane properties. We further characterize the evolution of V. splendidus membrane fluidity during different growth stages and relate it to fatty acid chain composition. Our results show larger membrane fluidity during the stationary growth phase compared to the exponential growth phase under labelling conditions - an information to take into account for future in vivo SS-NMR studies. Our lipid deuteration protocol optimized for V. splendidus is likely applicable other microorganisms for in vivo NMR studies. Keywords : In-cell NMR ; Deuterium ; Isotopic labelling ; Membrane fluidity ; Fatty acids ; Magic-angle spinning

Escherichia coli as host for membrane protein structure determination: a global analysis

Hattab G, Warschawski DE, Moncoq K, Miroux B. Escherichia coli as host for membrane protein structure determination: a global analysis. Scientific Reports. 2015;5(1): 12097.The structural biology of membrane proteins (MP) is hampered by the difficulty in producing and purifying them. A comprehensive analysis of protein databases revealed that 213 unique membrane protein structures have been obtained after production of the target protein in E. coli. The primary expression system used was the one based on the T7 RNA polymerase, followed by the arabinose and T5 promoter based expression systems. The C41λ(DE3) and C43λ(DE3) bacterial mutant hosts have contributed to 28% of non E. coli membrane protein structures. A large scale analysis of expression protocols demonstrated a preference for a combination of bacterial host-vector together with a bimodal distribution of induction temperature and of inducer concentration. Altogether our analysis provides a set of rules for the optimal use of bacterial expression systems in membrane protein production

Unambiguous Ex Situ and in Cell 2D ¹³C Solid-State NMR Characterization of Starch and Its Constituents

Starch is the most abundant energy storage molecule in plants and is an essential part of the human diet. This glucose polymer is composed of amorphous and crystalline domains in different forms (A and B types) with specific physicochemical properties that determine its bioavailability for an organism, as well as its value in the food industry. Using two-dimensional (2D) high resolution solid-state nuclear magnetic resonance (SS-NMR) on 13C-labelled starches that were obtained from Chlamydomonas reinhardtii microalgae, we established a complete and unambiguous assignment for starch and its constituents (amylopectin and amylose) in the two crystalline forms and in the amorphous state. We also assigned so far unreported non-reducing end groups and assessed starch chain length, crystallinity and amylose content. Starch was then characterized in situ, i.e., by 13C solid-state NMR of intact microalgal cells. Our in-cell methodology also enabled the identification of the effect of nitrogen starvation on starch metabolism. This work shows how solid-state NMR can enable the identification of starch structure, chemical modifications and biosynthesis in situ in intact microorganisms, eliminating time consuming and potentially altering purification steps

Study by NMR-based metabolomics of the impact of the toxic dinoflagellate Alexandrium catanela on blue mussel, Mytilus edulis, metabolism (NMR spectra)

NMR spectra (NOESY, COSY, TOCSY) of M. edulis (blue mussel) hemocytes and plasma after an exposition of 120h to the toxic algea Alexandrium catanela The spectra were aquired with a Bruker spectrometer operating at 600MHz equiped with a room temperature TXI probe.This project was undertaken with the financial support of the Government of Canada, specifically by a research grant from Environment and Climate Change Canada provided to RT's (FDE-CA-2017i002) and by the Ressources Aquatiques Québec research network (Fonds de Recherche du Québec – Nature et Technologies, no. 2014-RS-171172)

Perfil de la infraestructura logística de Colombia

En el siguiente documento se presenta una caracterización detallada sobre la situación de la infraestructura logística de Colombia, partiendo del estado en el cual se encuentra a nivel nacional y luego evidenciando, en lo posible, las ventajas y retos que existen con respecto a Latino América y el mundo. Actualmente Colombia presenta un rezago en su infraestructura, sus redes viales no son suficientes ni aptas para las operaciones de las empresas, las líneas férreas son obsoletas, las zonas portuarias no logran tener interconexiones óptimas con el centro del país y la capacidad de las terminales aéreas son un factor de retraso en actividades comerciales. Así mismo, la presencia de las zonas francas y de los parques industriales es menor con respecto a la importancia que estos tienen para el comercio nacional e internacional. Se revisarán cada uno de los modos de transporte: Red vial, red ferroviaria, transporte marítimo, red fluvial y sistema aéreo, en conjunto con la infraestructura que requiere cada uno para lograr ser eficientes y competitivos. Para cada modo se exponen sus antecedentes, la infraestructura, los equipos y tipos de cargas manejados en el país, los problemas y retos que se deben enfrentar, así como los aspectos legales que lo rodean y los planes a futuro. De igual forma, es importante estudiar las zonas francas y parques industriales como modos de apoyo y facilitadores para llevar a cabo el comercio de manera más efectiva y competitiva. Se presentará, al igual que en los modos de transporte, un resumen de los antecedentes, los problemas y retos más determinantes, la normativa legal que los acoge y los planes y políticas establecidos para los próximos años. Como parte importante del tránsito de mercancías para el comercio entre naciones, la zona sur del país es caracterizada para dar un diagnóstico actual de su infraestructura y de cómo esta afecta las operaciones. Finalmente, se pretende dar un panorama del cumplimiento de los proyectos establecidos por el gobierno en su Política Nacional Logística y en las políticas relacionadas con infraestructura y transporte, completando así, el diagnóstico de la infraestructura logística de Colombia. xii Lo anterior pretende evidenciar el desfase que existe entre el crecimiento económico y la infraestructura logística colombiana, que en conjunto con los sistemas de transporte y los medios de apoyo al comercio, resulta en una pérdida de competitividad del país a nivel internacional.The following document presents a detailed description of the current situation of the logistical infrastructure in Colombia, based on the state in which is currently found on the country, and then highlighting, where possible, the challenges and advantages on which Colombia stands against Latin America and the world. Currently Colombia presents a lag in its infrastructure; its roads are neither sufficient nor suited for the normal operation of any company, the rail roads are obsolete, the sea ports are unable to connect with the center of the county and the capacity of the airports constitutes the main delay factor in all commercial activities. Likewise, the presence of free trade zones and industrial parks is smaller regarding the importance that these have for domestic and international trade. Each of the different modes of transportation is going to be reviewed: roads network, railways, sea ports, the fluvial network and air transport, along with the infrastructure that each of them requires in order to be efficient and competitive. For each of the different modes, their background, infrastructure, equipment and types of cargo handled in the country, problems and challenges that need to be overcome, as well as the legal issues surrounding them along with their future corresponding plans, are going to be discussed. Likewise, it is important to study the free trade zones and industrial parks, as support systems and facilitators in order to carry out businesses more effectively and competitively. It will be presented as well as with the other modes of transportation, a summary of their background, the problems and challenges that they face, the legal regulations and the policies established for the coming years. Due to the importance that the south region of the country has in international trade, a characterization and diagnosis of its current infrastructure is presented, as well as how these issues are affecting normal day to day operations. Finally it is intended to give an overview of the implementation of the projects established by the government in its National Logistics Policy and policies related to the transportation infrastructure, completing thus the diagnosis of the logistical infrastructure of Colombia. xiv All of the above aims to show the gap between the economic growth and the logistical infrastructure in the country, that along with the transportation systems and other means that support trade activities, results in a loss of competitiveness of the country in an international level