Hydrogen/deuterium exchange-mass spectrometry of integral membrane proteins in native-like environments: current scenario and the way forward.

Integral membrane proteins (IMPs) perform a range of diverse functions and their dysfunction underlies numerous pathological conditions. Consequently, IMPs constitute most drug targets, and the elucidation of their mechanism of action has become an intense field of research. Historically, IMP studies have relied on their extraction from membranes using detergents, which have the potential to perturbate their structure and dynamics. To circumnavigate this issue, an array of membrane mimetics has been developed that aim to reconstitute IMPs into native-like lipid environments that more accurately represent the biological membrane. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a versatile tool for probing protein dynamics in solution. The continued development of HDX-MS methodology has allowed practitioners to investigate IMPs using increasingly native-like membrane mimetics, and even pushing the study of IMPs into the in vivo cellular environment. Consequently, HDX-MS has come of age and is playing an ever-increasingly important role in the IMP structural biologist toolkit. In the present mini-review, we discuss the evolution of membrane mimetics in the HDX-MS context, focusing on seminal publications and recent innovations that have led to this point. We also discuss state-of-the-art methodological and instrumental advancements that are likely to play a significant role in the generation of high-quality HDX-MS data of IMPs in the future.

Structural Insights into the Catalytic Cycle of a Bacterial Multidrug ABC Efflux Pump.

ABC ("ATP-Binding Cassette") transporters of the type IV subfamily consist of exporters involved in the efflux of many compounds, notably those capable to confer multidrug resistance like the mammalian P-glycoprotein or the bacterial transporter BmrA. They function according to an alternating access mechanism between inward-facing (IF) and outward-facing (OF) conformations, but the extent of physical separation between the two nucleotide-binding domains (NBDs) in different states is still unsettled. Small Angle Neutron Scattering and hydrogen/deuterium exchange coupled to mass spectrometry were used to highlight different conformational states of BmrA during its ATPase cycle. In particular, mutation of the conserved Lysine residue of the Walker-A motif (K380A) captures BmrA in an ATP-bound IF conformation prior to NBD closure. While in the transition-like state induced by vanadate wild-type BmrA is mainly in an OF conformation, the transporter populates only IF conformations in either the apo state or in the presence of ADP/Mg. Importantly, in this post-hydrolytic step, distances between the two NBDs of BmrA seem to be more separated than in the apo state, but they remain shorter than the widest opening found in the related MsbA transporter. Overall, our results highlight the main steps of the catalytic cycle of a homodimeric bacterial multidrug transporter and underline structural and functional commonalities as well as oddities among the type IV subfamily of ABC transporters.

Substrate-bound and substrate-free outward-facing structures of a multidrug ABC exporter.

Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg2+-bound outward-facing conformations of the Bacillus subtilis (homodimeric) BmrA by x-ray crystallography and single-particle cryo-electron microscopy (EM) in detergent solution, one of them with rhodamine 6G (R6G), a substrate exported by BmrA when overexpressed in B. subtilis. Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1-2 of one monomer and TM5'-6' of the other. They induce a rearrangement of TM1-2, highlighting a local flexibility that we confirmed by hydrogen/deuterium exchange and molecular dynamics simulations. In the absence of R6G, simulations show a fast postrelease occlusion of the cavity driven by hydrophobicity, while when present, R6G can move within the cavity, maintaining it open.

Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain.

Overexpression of correctly folded membrane proteins is a fundamental prerequisite for functional and structural studies. One of the most commonly used expression systems for the production of membrane proteins is Escherichia coli. While misfolded proteins typically aggregate and form inclusions bodies, membrane proteins that are addressed to the membrane and extractable by detergents are generally assumed to be properly folded. Accordingly, GFP fusion strategy is often used as a fluorescent proxy to monitor their expression and folding quality. Here we investigated the functionality of two different multidrug ABC transporters, the homodimer BmrA from Bacillus subtilis and the heterodimer PatA/PatB from Streptococcus pneumoniae, when produced in several E. coli strains with T7 expression system. Strikingly, while strong expression in the membrane of several strains could be achieved, we observed drastic differences in the functionality of these proteins. Moreover, we observed a general trend in which mild detergents mainly extract the population of active transporters, whereas a harsher detergent like Fos-choline 12 could solubilize transporters irrespective of their functionality. Our results suggest that the amount of T7 RNA polymerase transcripts may indirectly but notably impact the structure and activity of overexpressed membrane proteins, and advise caution when using GFP fusion strategy.

Assemblies of lauryl maltose neopentyl glycol (LMNG) and LMNG-solubilized membrane proteins.

Laurylmaltose neopentylglycol (LMNG) bears two linked hydrophobic chains of equal length and two hydrophilic maltoside groups. It arouses a strong interest in the field of membrane protein biochemistry, since it was shown to efficiently solubilize and stabilize membrane proteins often better than the commonly used dodecylmaltopyranoside (DDM), and to allow structure determination of some challenging membrane proteins. However, LMNG was described to form large micelles, which could be unfavorable for structural purposes. We thus investigated its auto-assemblies and the association state of different membrane proteins solubilized in LMNG by analytical ultracentrifugation, size exclusion chromatography coupled to light scattering, centrifugation on sucrose gradient and/or small angle scattering. At high concentrations (in the mM range), LMNG forms long rods, and it stabilized the membrane proteins investigated herein, i.e. a bacterial multidrug transporter, BmrA; a prokaryotic analogous of the eukaryotic NADPH oxidases, SpNOX; an E. coli outer membrane transporter, FhuA; and the halobacterial bacteriorhodopsin, bR. BmrA, in the Apo and the vanadate-inhibited forms showed reduced kinetics of limited proteolysis in LMNG compared to DDM. Both SpNOX and BmrA display an increased specific activity in LMNG compared to DDM. The four proteins form LMNG complexes with their usual quaternary structure and with usual amount of bound detergent. No heterogeneous complexes related to the large micelle size of LMNG alone were observed. In conditions where LMNG forms assemblies of large size, FhuA crystals diffracting to 4.0 Šwere obtained by vapor diffusion. LMNG large micelle size thus does not preclude membrane protein homogeneity and crystallization.

ThemeDelta: Dynamic Segmentations over Temporal Topic Models.

We present ThemeDelta, a visual analytics system for extracting and visualizing temporal trends, clustering, and reorganization in time-indexed textual datasets. ThemeDelta is supported by a dynamic temporal segmentation algorithm that integrates with topic modeling algorithms to identify change points where significant shifts in topics occur. This algorithm detects not only the clustering and associations of keywords in a time period, but also their convergence into topics (groups of keywords) that may later diverge into new groups. The visual representation of ThemeDelta uses sinuous, variable-width lines to show this evolution on a timeline, utilizing color for categories, and line width for keyword strength. We demonstrate how interaction with ThemeDelta helps capture the rise and fall of topics by analyzing archives of historical newspapers, of U.S. presidential campaign speeches, and of social messages collected through iNeighbors, a web-based social website. ThemeDelta is evaluated using a qualitative expert user study involving three researchers from rhetoric and history using the historical newspapers corpus.

Stack zooming for multifocus interaction in skewed-aspect visual spaces.

Many 2D visual spaces have a virtually one-dimensional nature with very high aspect ratio between the dimensions: examples include time-series data, multimedia data such as sound or video, text documents, and bipartite graphs. Common among these is that the space can become very large, e.g., temperature measurements could span a long time period, surveillance video could cover entire days or weeks, and documents can have thousands of pages. Many analysis tasks for such spaces require several foci while retaining context and distance awareness. In this extended version of our IEEE PacificVis 2010 paper, we introduce a method for supporting this kind of multifocus interaction that we call stack zooming. The approach is based on building hierarchies of 1D strips stacked on top of each other, where each subsequent stack represents a higher zoom level, and sibling strips represent branches in the exploration. Correlation graphics show the relation between stacks and strips of different levels, providing context and distance awareness for the foci. The zoom hierarchies can also be used as graphical histories and for communicating insights to stakeholders and can be further extended with annotation and integrated statistics.

Evaluating the Role of Time in Investigative Analysis of Document Collections.

Time is a universal and essential aspect of data in any investigative analysis. It helps analysts establish causality, build storylines from evidence, and reject infeasible hypotheses. For this reason, many investigative analysis tools provide visual representations designed for making sense of temporal data. However, the field of visual analytics still needs more evidence explaining how temporal visualization actually aids the analysis process, as well as design recommendations for how to build these visualizations. To fill this gap, we conducted an insight-based qualitative study to investigate the influence of temporal visualization on investigative analysis. We found that visualizing temporal information helped participants externalize chains of events. Another contribution of our work is the lightweight evaluation approach used to collect, visualize, and analyze insight.

Graphical perception of multiple time series.

Line graphs have been the visualization of choice for temporal data ever since the days of William Playfair (1759-1823), but realistic temporal analysis tasks often include multiple simultaneous time series. In this work, we explore user performance for comparison, slope, and discrimination tasks for different line graph techniques involving multiple time series. Our results show that techniques that create separate charts for each time series--such as small multiples and horizon graphs--are generally more efficient for comparisons across time series with a large visual span. On the other hand, shared-space techniques--like standard line graphs--are typically more efficient for comparisons over smaller visual spans where the impact of overlap and clutter is reduced.