scholarly journals New method for high-throughput measurements of viscosity in submicrometer-sized membrane systems

2019 ◽  
Author(s):  
Grzegorz Chwastek ◽  
Eugene P. Petrov ◽  
James Peter Sáenz
Keyword(s):  
High Throughput ◽  
Lipid Membranes ◽  
High Throughput Screening ◽  
Lipid Membrane ◽  
Fluorescence Emission ◽  
Lipid Phase ◽  
Bacterial Cells ◽  
Membrane Adaptation ◽  
Mycoplasma Mycoides ◽  
Good Agreement

AbstractIn order to unravel the underlying principles of membrane adaptation in small systems like bacterial cells, robust approaches to characterize membrane fluidity are needed. Currently available relevant methods require advanced instrumentation and are not suitable for high throughput settings needed to elucidate the biochemical pathways involved in adaptation. We developed a fast, robust, and financially accessible quantitative method to measure microviscosity of lipid membranes in bulk suspension using a commercially available plate reader. Our approach, which is suitable for high-throughput screening, is based on the simultaneous measurements of absorbance and fluorescence emission of a viscosity-sensitive fluorescent dye DCVJ incorporated into a lipid membrane. We validated our method using artificial membranes with various lipid compositions over a range of temperatures and observed values that were in good agreement with previously published results. Using our approach, we were able to detect a lipid phase transition in the ruminant pathogen Mycoplasma mycoides.

scholarly journals Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Emma Barahona ◽  
Emilio Jiménez-Vicente ◽  
Luis M. Rubio
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Rhodobacter Capsulatus ◽  
Random Mutagenesis ◽  
Fluorescence Emission ◽  
Inducible Promoter ◽  
Hydrogen Gas ◽  
Photosynthetic Organisms ◽  
Fe Protein ◽  
Protein Variants

Abstract When produced biologically, especially by photosynthetic organisms, hydrogen gas (H2) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H2-producing enzymes is the absence of methods for the high-throughput screening of H2 production in biological systems. Here, we re-engineered the natural H2 sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H2. A lacZ gene was placed under the control of the hupA H2-inducible promoter in a strain lacking the uptake hydrogenase and the nifH nitrogenase gene. This system was then used in combination with fluorescence-activated cell sorting flow cytometry to screen large libraries of nitrogenase Fe protein variants generated by random mutagenesis. Exact correlation between fluorescence emission and H2 production levels was found for all automatically selected strains. One of the selected H2-overproducing Fe protein variants lacked 40% of the wild-type amino acid sequence, a surprising finding for a protein that is highly conserved in nature. We propose that this method has great potential to improve microbial H2 production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.

scholarly journals Colloidal Aggregate Detection by Rapid Fluorescence Measurement of Liquid Surface Curvature Changes in Multiwell Plates

2007 ◽  
Vol 12 (7) ◽  
pp. 966-971 ◽  
Author(s):  
Lifeng Cai ◽  
Miriam Gochin
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Dynamic Range ◽  
Liquid Surface ◽  
Enzyme Inhibitor ◽  
Fluorescence Emission ◽  
Fluorescence Measurement ◽  
Screening Assay ◽  
Colloidal Aggregate ◽  
Multiwell Plates

A simple fluorescence method is reported for the detection of colloidal aggregate formation in solution, with specific applications to determine the critical micelle concentration (CMC) of surfactants and detect small-molecule promiscuous inhibitors. The method exploits the meniscus curvature changes in high-density multiwell plates associated with colloidal changes in solution. The shape of the meniscus has a significant effect on fluorescence intensity when detected using a top-read fluorescence plate reader because of the effect of total internal reflection on fluorescence emission through a curved liquid surface. A dynamic range of 60% is calculated and observed and is measured with a relative sensitivity of 2%. Facile determination of the CMC of a variety of surfactants is demonstrated, as well as a screening assay for aggregate forming properties of small drug-like compounds, a common cause of promiscuous inhibition in high-throughput screening (HTS) enzyme inhibitor assays. Our preliminary results show a potential HTS assay with Z′ factor of 0.76, with good separation between aggregating and nonaggregating small molecules. The method combines the high sensitivity and universality of classic surface tension methods with simplicity and high-throughput determination, enabling facile detection of molecular interactions involving a change in liquid or solid surface character. ( Journal of Biomolecular Screening 2007:966-971)

scholarly journals Membrane thickness, lipid phase and sterol type are determining factors in the permeability of membranes to small solutes

2021 ◽  
Author(s):  
Jacopo Frallicciardi ◽  
Josef Melcr ◽  
Pareskevi Siginou ◽  
Siewert Marrink ◽  
Bert Poolman
Keyword(s):  
Lipid Membranes ◽  
Lipid Phase ◽  
Membrane Thickness ◽  
Bacterial Cells ◽  
Concentration Gradients ◽  
Kinetic Measurements ◽  
Systematic Analysis ◽  
Liquid Ordered ◽  
Dynamics Simulations

Abstract Cell membranes provide a selective semi-permeable barrier to the passive transport of molecules. This property differs greatly between organisms. While the cytoplasmic membrane of bacterial cells is highly permeable for weak acids and glycerol, yeasts can maintain large concentration gradients. Here we show that such differences can arise from the physical state of the plasma membrane. By combining stopped-flow kinetic measurements with molecular dynamics simulations, we performed a systematic analysis of the permeability through synthetic lipid membranes to obtain detailed molecular insight into the permeation mechanisms. While membrane thickness is an important parameter for the permeability through fluid membranes, the largest differences occur when the membranes transit from the liquid-disordered to liquid-ordered and/or to gel state. By comparing our results with in vivo measurements from yeast, we conclude that the yeast membrane exists in a highly ordered and rigid state, which is comparable to synthetic saturated DPPC-sterol membranes.

scholarly journals Matrix-Based Activity Pattern Classification as a Novel Method for the Characterization of Enzyme Inhibitors Derived from High-Throughput Screening

2016 ◽  
Vol 21 (10) ◽  
pp. 1075-1089 ◽  
Author(s):  
Douglas S. Auld ◽  
Marta Jimenez ◽  
Kimberley Yue ◽  
Scott Busby ◽  
Yu-Chi Chen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Enzyme Inhibitors ◽  
Classification Method ◽  
Shift Method ◽  
Lead Selection ◽  
Small Matrix ◽  
Novel Method ◽  
Good Agreement

One of the central questions in the characterization of enzyme inhibitors is determining the mode of inhibition (MOI). Classically, this is done with a number of low-throughput methods in which inhibition models are fitted to the data. The ability to rapidly characterize the MOI for inhibitors arising from high-throughput screening in which hundreds to thousands of primary inhibitors may need to be characterized would greatly help in lead selection efforts. Here we describe a novel method for determining the MOI of a compound without the need for curve fitting of the enzyme inhibition data. We provide experimental data to demonstrate the utility of this new high-throughput MOI classification method based on nonparametric analysis of the activity derived from a small matrix of substrate and inhibitor concentrations (e.g., from a 4S × 4I matrix). Lists of inhibitors from four different enzyme assays are studied, and the results are compared with the previously described IC50-shift method for MOI classification. The MOI results from this method are in good agreement with the known MOI and compare favorably with those from the IC50-shift method. In addition, we discuss some advantages and limitations of the method and provide recommendations for utilization of this MOI classification method.

scholarly journals Mimicking the Mammalian Plasma Membrane: An Overview of Lipid Membrane Models for Biophysical Studies

Biomimetics ◽  
2020 ◽  
Vol 6 (1) ◽  
pp. 3
Author(s):  
Alessandra Luchini ◽  
Giuseppe Vitiello
Keyword(s):  
Plasma Membrane ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Cell Membranes ◽  
Lipid Membrane ◽  
Lipid Phase ◽  
Chemical Information ◽  
Lipid Species ◽  
Membrane Models ◽  
The Impact

Cell membranes are very complex biological systems including a large variety of lipids and proteins. Therefore, they are difficult to extract and directly investigate with biophysical methods. For many decades, the characterization of simpler biomimetic lipid membranes, which contain only a few lipid species, provided important physico-chemical information on the most abundant lipid species in cell membranes. These studies described physical and chemical properties that are most likely similar to those of real cell membranes. Indeed, biomimetic lipid membranes can be easily prepared in the lab and are compatible with multiple biophysical techniques. Lipid phase transitions, the bilayer structure, the impact of cholesterol on the structure and dynamics of lipid bilayers, and the selective recognition of target lipids by proteins, peptides, and drugs are all examples of the detailed information about cell membranes obtained by the investigation of biomimetic lipid membranes. This review focuses specifically on the advances that were achieved during the last decade in the field of biomimetic lipid membranes mimicking the mammalian plasma membrane. In particular, we provide a description of the most common types of lipid membrane models used for biophysical characterization, i.e., lipid membranes in solution and on surfaces, as well as recent examples of their applications for the investigation of protein-lipid and drug-lipid interactions. Altogether, promising directions for future developments of biomimetic lipid membranes are the further implementation of natural lipid mixtures for the development of more biologically relevant lipid membranes, as well as the development of sample preparation protocols that enable the incorporation of membrane proteins in the biomimetic lipid membranes.

Development and Validation of a High-Throughput Screening Assay for the Hepatitis C Virus p7 Viroporin

2011 ◽  
Vol 16 (3) ◽  
pp. 363-369 ◽  
Author(s):  
Christian Gervais ◽  
Florence Dô ◽  
Ariane Cantin ◽  
George Kukolj ◽  
Peter W. White ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Lipid Membrane ◽  
Robust Statistics ◽  
Phospholipid Composition ◽  
Reversed Phase ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Fed Batch Fermentation ◽  
Dye Permeability

The HCV p7 protein is not involved in viral RNA replication but is essential for production of infectious virus. Based on its putative ion channel activity, p7 belongs to a family of viral proteins known as viroporins that oligomerize after insertion into a lipid membrane. To screen for compounds capable of interfering with p7 channel function, a low-throughput liposome-based fluorescent dye permeability assay was modified and converted to a robust high-throughput screening assay. Escherichia coli expressing recombinant p7 were grown in high-density fed-batch fermentation followed by a detergent-free purification using a combination of affinity and reversed-phase chromatography. The phospholipid composition of the liposomes was optimized for both p7 recognition and long-term stability. A counterscreen was developed using the melittin channel-forming peptide to eliminate nonspecific screening hits. The p7 liposome-based assay displayed robust statistics (Z′ > 0.75), and sensitivity to inhibition was confirmed using known inhibitors.

scholarly journals Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells

2016 ◽  
Vol 22 (3) ◽  
pp. 250-261 ◽  
Author(s):  
Tory M. Schaaf ◽  
Kurt C. Peterson ◽  
Benjamin D. Grant ◽  
David D. Thomas ◽  
Gregory D. Gillispie
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Structural Changes ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Resonance Energy ◽  
Living Cells ◽  
Spectral Unmixing ◽  
Fluorescence Emission Spectrum ◽  
Plate Reader

We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

High Pressure X-ray Studies of Lipid Membranes and Lipid Phase Transitions

2014 ◽  
Vol 228 (10-12) ◽  
Author(s):  
Nicholas J. Brooks ◽  
John M. Seddon
Keyword(s):  
High Pressure ◽  
Phase Transitions ◽  
Lipid Membranes ◽  
Soft Matter ◽  
Lipid Membrane ◽  
Lipid Phase ◽  
Pressure Jump ◽  
X Ray ◽  
Lipid Phase Transitions ◽  
X Ray Scattering

AbstractHydrostatic pressure has dramatic effects on biomembrane structure and stability and is a key thermodynamic parameter in the context of the biology of deep sea organisms. Furthermore, high-pressure and pressure-jump studies are very useful tools in biophysics and biotechnology, where they can be used to study the mechanism and kinetics of lipid phase transitions, biomolecular transformations, and protein folding/unfolding. Here, we first give an overview of the technology currently available for X-ray scattering studies of soft matter systems under pressure. We then illustrate the use of this technology to study a variety of lipid membrane systems.

scholarly journals Assessing the use of ellipsoidal microparticles for determining lipid membrane viscosity

2021 ◽  
Author(s):  
Philip E. Jahl ◽  
Raghuveer Parthasarathy
Keyword(s):  
Lipid Membranes ◽  
Lipid Membrane ◽  
Translational Diffusion ◽  
Lipid Domains ◽  
Giant Vesicles ◽  
Membrane Viscosity ◽  
Cellular Processes ◽  
Wide Range ◽  
Chain Lengths ◽  
Good Agreement

The viscosity of lipid membranes sets the timescales of membrane-associated flows and therefore influences the dynamics of a wide range of cellular processes. Techniques to measure membrane viscosity remain sparse, however, and reported measurements to date, even of similar systems, give viscosity values that span orders of magnitude. To address this, we improve a method based on measuring both the rotational and translational diffusion of membrane-anchored microparticles and apply this approach and one based on tracking the motion of phase-separated lipid domains to the same system of phase-separated giant vesicles. We find good agreement between the two methods, with inferred viscosities within a factor of two of each other. Our technique uses ellipsoidal microparticles, and we show that the extraction of physically meaningful viscosity values from their motion requires consideration of their anisotropic shape. The validation of our method on phase-separated membranes makes possible its application to other systems, which we demonstrate by measuring the viscosity of bilayers composed of lipids with different chain lengths ranging from 14 to 20 carbon atoms, revealing a very weak dependence of two-dimensional viscosity on lipid size. The experimental and analysis methods described here should be generally applicable to a variety of membrane systems, both reconstituted and cellular.

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