Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers : Nanopore Engineering and Characterization

Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein's insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal's DNA-ratchet function

High-throughput expression and purification of human solute carriers for structural and biochemical studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening

High-resolution spatio-temporal analyses of drought episodes in the western Mediterranean basin (Spanish mainland, Iberian Peninsula)

12 Pags.- 6 Figs.- 1 Tabl. © The Author(s) 2018. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The purpose of this research was to identify major drought events on the Spanish mainland between 1961 and 2014 by means of two drought indices, and analyze the spatial propagation of drought conditions. The indices applied were the standardized precipitation index (SPI) and the standardized evaporation precipitation index (SPEI). The first was calculated as standardized anomalies of precipitation at various temporal intervals, while the second examined the climatic balance normalized at monthly scale, incorporating the relationship between precipitation and the atmospheric water demand. The daily meteorological data from Spanish Meteorological Archives (AEMet) were used in performing the analyses. Within the framework of the DESEMON project, original data were converted into a high spatial resolution grid (1.1 km2) following exhaustive quality control. Values of both indices were calculated on a weekly scale and different timescales (12, 24 and 36 months). The results show that during the first half of the study period, the SPI usually returned a higher identification of drought areas, while the reverse was true from the 1990s, suggesting that the effect from atmospheric evaporative demand could have increased. The temporal propagation from 12- to 24-month and 36-month timescales analyzed in the paper seems to be a far from straightforward phenomenon that does not follow a simple rule of time lag, because events at different temporal scales can overlap in time and space. Spatially, the propagation of drought events affecting more than 25% of the total land indicates the existence of various spatial gradients of drought propagation, mostly east–west or west–east, but also north–south have been found. No generalized episodes were found with a radial pattern, i.e., from inland to the coast.This work is supported by the projects CGL2014-52135-C03-01, CGL2014-52135-C3-3-R and PCIN-2015-220, financed by the Spanish Government, Ministry of the Economy and Finance and FEDER, IMDROFLOOD financed by the Water Works 2014 co-funded call of the European Commission, INDECIS, financed by the ERA-NET Cofund for Climate Services of the European Research Area for Climate Services, and the Regional Government of Aragón DGA-FSE (Grupo de Investigación Consolidado ‘Clima, Agua, Cambio Global y Sistemas Naturales’). Celia Salinas is a FPI-PhD student supported by the Ministry of the Economy and Finance. Miquel Tomas-Burguera was supported by the predoctoral FPU program 2013 (Ministry of Education, Culture and Sport).Peer reviewe