An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (www.mpdb.tcd.ie). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.
25 Related JoVE Articles!
Crystallization of Membrane Proteins in Lipidic Mesophases
Institutions: The Scripps Research Institute.
Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso
or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso
crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.
Structural Biology, Issue 49, membrane protein, lipidic cubic phase, crystallization, Fluorescence recovery after photobleaching (FRAP) , G protein-coupled receptors
Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases
Institutions: Trinity College Dublin.
A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso
method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso
crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.
Biochemistry, Issue 45, membrane protein, in meso, membrane, crystallization, lipidic mesophases, manual
Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties
Institutions: Weill Cornell Medical College.
Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12
. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+
that has been successfully used in other fluorescence quenching assays 5,13
permeates the lipid bilayer slowly 8
but passes readily through conducting gramicidin channels 1,14
. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12
Microbiology, Issue 44, membrane properties, bilayer properties, gramicidin, fluorescence quenching, high throughput drug screening
A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry
Institutions: University of Alberta, Universidad Nacional Autónoma de México, Universidad Nacional Autónoma de México.
Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-κB and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.
Immunology, Issue 74, Biochemistry, Infection, Cellular Biology, Molecular Biology, Medicine, Neutrophils, Neutrophil, Monocyte, PMN, NF- κB, ERK, integrin, Signal Transduction, inflammation, flow cytometry, immunolabeling, nuclear factors, cytokines, cells, assay
Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities
Institutions: Université de Strasbourg.
Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols.
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.
Neuroscience, Issue 89, mice, nociception, tail immersion test, tail pressure test, morphine, analgesia, opioid-induced hyperalgesia, tolerance
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Institutions: The University of Chicago Medical Center, The University of Chicago Medical Center.
Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro
using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro
preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers.
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo
counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure
neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic
SD immune signaling.
Neuroscience, Issue 52, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy
Institutions: Carnegie Mellon University.
Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.
Cellular Biology, Issue 92, Endocytosis, TIRF, total internal reflection fluorescence microscopy, clathrin, arrestin, receptors, live-cell microscopy, clathrin-mediated endocytosis
Quantitative Measurement of the Immune Response and Sleep in Drosophila
Institutions: University of Pennsylvania Perelman School of Medicine.
A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1
and has also recently been described in Drosophila melanogaster2
. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4
. However, experimental evidence that supports this hypothesis is limited4
, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster
. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NFκB, a key transcription factor that is central to the innate immune response in Drosophila
. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5
. Real-time monitoring of NFκB activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila
in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.
Immunology, Issue 70, Neuroscience, Medicine, Physiology, Pathology, Microbiology, immune response, sleep, Drosophila, infection, bacteria, luciferase reporter assay, animal model
Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood
Institutions: Yale University School of Medicine .
Individual variations in immune status determine responses to infection and contribute to disease severity and outcome. Aging is associated with an increased susceptibility to viral and bacterial infections and decreased responsiveness to vaccines with a well-documented decline in humoral as well as cell-mediated immune responses1,2
. We have recently assessed the effects of aging on Toll-like receptors (TLRs), key components of the innate immune system that detect microbial infection and trigger antimicrobial host defense responses3
. In a large cohort of healthy human donors, we showed that peripheral blood monocytes from the elderly have decreased expression and function of certain TLRs4
and similar reduced TLR levels and signaling responses in dendritic cells (DCs), antigen-presenting cells that are pivotal in the linkage between innate and adaptive immunity5
. We have shown dysregulation of TLR3 in macrophages and lower production of IFN by DCs from elderly donors in response to infection with West Nile virus6,7
Paramount to our understanding of immunosenescence and to therapeutic intervention is a detailed understanding of specific cell types responding and the mechanism(s) of signal transduction. Traditional studies of immune responses through imaging of primary cells and surveying cell markers by FACS or immunoblot have advanced our understanding significantly, however, these studies are generally limited technically by the small sample volume available from patients and the inability to conduct complex laboratory techniques on multiple human samples. ImageStream combines quantitative flow cytometry with simultaneous high-resolution digital imaging and thus facilitates investigation in multiple cell populations contemporaneously for an efficient capture of patient susceptibility. Here we demonstrate the use of ImageStream in DCs to assess TLR7/8 activation-mediated increases in phosphorylation and nuclear translocation of a key transcription factor, NF-κB, which initiates transcription of numerous genes that are critical for immune responses8
. Using this technology, we have also recently demonstrated a previously unrecognized alteration of TLR5 signaling and the NF-κB pathway in monocytes from older donors that may contribute to altered immune responsiveness in aging9
Immunology, Issue 62, monocyte, dendritic cells, Toll-like receptors, fluorescent imaging, signaling, FACS, aging
Sputter Growth and Characterization of Metamagnetic B2-ordered FeRh Epilayers
Institutions: University of Leeds, University of Leeds, University of Edinburgh, Northeastern University, Northeastern University.
Chemically ordered alloys are useful in a variety of magnetic nanotechnologies. They are most conveniently prepared at an industrial scale using sputtering techniques. Here we describe a method for preparing epitaxial thin films of B2-ordered FeRh by sputter deposition onto single crystal MgO substrates. Deposition at a slow rate onto a heated substrate allows time for the adatoms to both settle into a lattice with a well-defined epitaxial relationship with the substrate and also to find their proper places in the Fe and Rh sublattices of the B2 structure. The structure is conveniently characterized with X-ray reflectometry and diffraction and can be visualised directly using transmission electron micrograph cross-sections. B2-ordered FeRh exhibits an unusual metamagnetic phase transition: the ground state is antiferromagnetic but the alloy transforms into a ferromagnet on heating with a typical transition temperature of about 380 K. This is accompanied by a 1% volume expansion of the unit cell: isotropic in bulk, but laterally clamped in an epilayer. The presence of the antiferromagnetic ground state and the associated first order phase transition is very sensitive to the correct equiatomic stoichiometry and proper B2 ordering, and so is a convenient means to demonstrate the quality of the layers that can be deposited with this approach. We also give some examples of the various techniques by which the change in phase can be detected.
Physics, Issue 80, Sputtering, epitaxial growth, magnetism, ordered alloys
High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method
Institutions: University of California Los Angeles , David Geffen School of Medicine, UCLA.
Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity.
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent′s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1
. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2
. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4
. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6
). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots.
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1)
. This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer′s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.
Molecular Biology, Issue 59, membrane proteins crystallization, bicelle, lipidic crystallization
Protein Crystallization for X-ray Crystallography
Institutions: Yale University.
Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.
Molecular Biology, Issue 47, protein crystallization, nucleic acid crystallization, vapor diffusion, X-ray crystallography, precipitant
Transmembrane Domain Oligomerization Propensity determined by ToxR Assay
Institutions: University of Colorado at Boulder.
The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven. In many cases membrane-spanning proteins have evolved highly sophisticated mechanisms of action.1-3
One way in which membrane proteins can modulate their structures and functions is by direct and specific contact of hydrophobic helices, forming structured transmembrane oligomers.4,5
Much recent work has focused on the distribution of amino acids preferentially found in the membrane environment in comparison to aqueous solution and the different intermolecular forces that drive protein association.6,7
Nevertheless, studies of molecular recognition at the transmembrane domain of proteins still lags behind those of water-soluble regions. A major hurdle remains: despite the remarkable specificity and affinity that transmembrane oligomerization can achieve,8
direct measurement of their association is challenging. Traditional methodologies applied to the study of integral membrane protein function can be hampered by the inherent insolubility of the sequences under examination. Biophysical insights gained from studying synthetic peptides representing transmembrane domains can provide useful structural insight. However, the biological relevance of the detergent micellar or liposome systems used in these studies to mimic cellular membranes is often questioned; do peptides adopt a native-like structure under these conditions and does their functional behaviour truly reflect the mode of action within a native membrane? In order to study the interactions of transmembrane sequences in natural phospholipid bilayers, the Langosch lab developed ToxR transcriptional reporter assays.9
The transmembrane domain of interest is expressed as a chimeric protein with maltose binding protein for location to the periplasm and ToxR to provide a report of the level of oligomerization (Figure 1).
In the last decade, several other groups (e.g. Engelman, DeGrado, Shai) further optimized and applied this ToxR reporter assay.10-13
The various ToxR assays have become a gold standard to test protein-protein interactions in cell membranes. We herein demonstrate a typical experimental operation conducted in our laboratory that primarily follows protocols developed by Langosch. This generally applicable method is useful for the analysis of transmembrane domain self-association in E. coli
, where β-galactosidase production is used to assess the TMD oligomerization propensity. Upon TMD-induced dimerization, ToxR binds to the ctx
promoter causing up-regulation of the LacZ
gene for β-galactosidase. A colorimetric readout is obtained by addition of ONPG to lyzed cells. Hydrolytic cleavage of ONPG by β-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP) (Figure 2).
Cellular Biology, Issue 51, Transmembrane domain, oligomerization, transcriptional reporter, ToxR, latent membrane protein-1
Designing a Bio-responsive Robot from DNA Origami
Institutions: Bar-Ilan University.
Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1
, they can be utilized in parallel computing2,3
, recognize and bind molecular or cellular targets4,5
, catalyze chemical reactions6,7
, and generate calculated responses in a biological system8,9
. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12
, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13
, and several variations on this theme have since been realized using various techniques11,12,14,15
. However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16
. In this technique, the folding of a long (>7,000 bases) single-stranded DNA 'scaffold'
is directed to a desired shape by hundreds of short complementary strands termed 'staples'
. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18
The current paper will focus on the caDNAno 2.0 software19
developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20
. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21
to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.
Bioengineering, Issue 77, Genetics, Biomedical Engineering, Molecular Biology, Medicine, Genomics, Nanotechnology, Nanomedicine, DNA origami, nanorobot, caDNAno, DNA, DNA Origami, nucleic acids, DNA structures, CAD, sequencing
Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications
Institutions: Purdue University, Eli Lilly and Company.
Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson's disease. Acute activation of Gαi/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro
and in vivo
following the chronic activation of several types of Gαi/o-linked receptors including D2
dopamine and μ opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gbγ signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384 well format would facilitate future studies. Using two D2
dopamine receptor cellular models (i.e
), we have converted our 48-well sensitization assay (>20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.
Bioengineering, Issue 83, adenylyl cyclase, cAMP, heterologous sensitization, superactivation, D2 dopamine, μ opioid, siRNA
3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache
Institutions: University of Michigan School of Dentistry, University of Michigan School of Dentistry, University of Michigan, University of Michigan, University of Michigan, University of Michigan.
A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration of specific neural processes in the central nervous system. However, information is lacking on the molecular impact of these changes, especially on the endogenous opioid system during migraine headaches, and neuronavigation through these changes has never been done. This study aimed to investigate, using a novel 3D immersive and interactive neuronavigation (3D-IIN) approach, the endogenous µ-opioid transmission in the brain during a migraine headache attack in vivo
. This is arguably one of the most central neuromechanisms associated with pain regulation, affecting multiple elements of the pain experience and analgesia. A 36 year-old female, who has been suffering with migraine for 10 years, was scanned in the typical headache (ictal) and nonheadache (interictal) migraine phases using Positron Emission Tomography (PET) with the selective radiotracer [11
C]carfentanil, which allowed us to measure µ-opioid receptor availability in the brain (non-displaceable binding potential - µOR BPND
). The short-life radiotracer was produced by a cyclotron and chemical synthesis apparatus on campus located in close proximity to the imaging facility. Both PET scans, interictal and ictal, were scheduled during separate mid-late follicular phases of the patient's menstrual cycle. During the ictal PET session her spontaneous headache attack reached severe intensity levels; progressing to nausea and vomiting at the end of the scan session. There were reductions in µOR BPND
in the pain-modulatory regions of the endogenous µ-opioid system during the ictal phase, including the cingulate cortex, nucleus accumbens (NAcc), thalamus (Thal), and periaqueductal gray matter (PAG); indicating that µORs were already occupied by endogenous opioids released in response to the ongoing pain. To our knowledge, this is the first time that changes in µOR BPND
during a migraine headache attack have been neuronavigated using a novel 3D approach. This method allows for interactive research and educational exploration of a migraine attack in an actual patient's neuroimaging dataset.
Medicine, Issue 88, μ-opioid, opiate, migraine, headache, pain, Positron Emission Tomography, molecular neuroimaging, 3D, neuronavigation
Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies
Institutions: University of Texas Southwestern Medical Center at Dallas.
To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.
Molecular Biology, Issue 77, Biochemistry, Genetics, Cellular Biology, Structural Biology, Biophysics, Membrane Lipids, Phospholipids, Carrier Proteins, Membrane Proteins, Micelles, Molecular Motor Proteins, life sciences, biochemistry, Amino Acids, Peptides, and Proteins, lipid-protein interaction, channel reconstitution, lipid-dependent gating, voltage-gated ion channel, conformation-specific ligands, lipids
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer
Institutions: Max Planck Institute for Psycholinguistics, Donders Institute for Brain, Cognition and Behaviour.
Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis
luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.
Cellular Biology, Issue 87, Protein-protein interactions, Bioluminescence Resonance Energy Transfer, Live cell, Transfection, Luciferase, Yellow Fluorescent Protein, Mutations
Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors
Institutions: University College London.
Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA
Rs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials.
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAA
Rs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other.
To elucidate the underlying molecular mechanisms, a novel in vitro
co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAA
R subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAA
R subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro
model system can be used to reproduce, at least in part, the in vivo
conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAA
Rs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.
Neuroscience, Issue 93, Developmental neuroscience, synaptogenesis, synaptic inhibition, co-culture, stable cell lines, GABAergic, medium spiny neurons, HEK 293 cell line
Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Institutions: The Molecular Foundry.
Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2
, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3
. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5
. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6
. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.
Environmental Sciences, Issue 90, small and asymmetric protein structure, electron microscopy, optimized negative staining
Using Microwave and Macroscopic Samples of Dielectric Solids to Study the Photonic Properties of Disordered Photonic Bandgap Materials
Institutions: San Francisco State University.
Recently, disordered photonic materials have been suggested as an alternative to periodic crystals for the formation of a complete photonic bandgap (PBG). In this article we will describe the methods for constructing and characterizing macroscopic disordered photonic structures using microwaves. The microwave regime offers the most convenient experimental sample size to build and test PBG media. Easily manipulated dielectric lattice components extend flexibility in building various 2D structures on top of pre-printed plastic templates. Once built, the structures could be quickly modified with point and line defects to make freeform waveguides and filters. Testing is done using a widely available Vector Network Analyzer and pairs of microwave horn antennas. Due to the scale invariance property of electromagnetic fields, the results we obtained in the microwave region can be directly applied to infrared and optical regions. Our approach is simple but delivers exciting new insight into the nature of light and disordered matter interaction.
Our representative results include the first experimental demonstration of the existence of a complete and isotropic PBG in a two-dimensional (2D) hyperuniform disordered dielectric structure. Additionally we demonstrate experimentally the ability of this novel photonic structure to guide electromagnetic waves (EM) through freeform waveguides of arbitrary shape.
Physics, Issue 91, optics and photonics, photonic crystals, photonic bandgap, hyperuniform, disordered media, waveguides
Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity
Institutions: Queen's University, Porter Neuroscience Research Center, National Institute of Advanced Industrial Science and Technology, The Hebrew University of Jerusalem.
Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a-
axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.
Chemistry, Issue 83, Materials, Life Sciences, Optics, antifreeze proteins, Ice adsorption, Fluorescent labeling, Ice lattice planes, ice-binding proteins, Single ice crystal
Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-κB Reporter Transgenic
Institutions: Vanderbilt University Medical Center, Vanderbilt University Medical Center, University of Hawaii at Hilo College of Pharmacy.
Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as pre-clinical models to test interventions. In these methods, we describe a technique in which lipopolysaccharide (LPS) is injected into the lactating mouse mammary gland via the nipple, effectively modeling mastitis, or inflammation, of the gland. This simulated infection results in increased nuclear factor kappa B (NF-κB) signaling, as visualized through bioluminescent imaging of an NF-κB luciferase reporter mouse1
Our ultimate goal in developing these methods was to study the inflammation associated with mastitis in the lactating gland, which often includes redness, swelling, and immune cell infiltration2,3
. Therefore, we were keenly aware that incision or any type of wounding of the skin, the nipple, or the gland in order to introduce the LPS could not be utilized in our methods since the approach would likely confound the read-out of inflammation. We also desired a straight-forward method that did not require specially made hand-drawn pipettes or the use of micromanipulators to hold these specialized tools in place. Thus, we determined to use a commercially available insulin syringe and to inject the agent into the mammary duct of an intact nipple. This method was successful and allowed us to study the inflammation associated with LPS injection without any additional effects overlaid by the process of injection. In addition, this method also utilized an NF-κB luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-κB signaling within the LPS-injected gland4
These methods are of interest to researchers of many disciplines who wish to model disease within the lactating mammary gland, as ultimately, the technique described here could be utilized for injection of a number of substances, and is not limited to only LPS.
Medicine, Issue 67, mastitis, intraductal injection, NF-kappaB, reporter transgenic, LPS, bioluminescent imaging, lactation
Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography
Institutions: Georgia Institute of Technology, RWTH Aachen University, Georgia Institute of Technology.
Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.
Cellular Biology, Issue 44, membrane protein, structure, two-dimensional crystallization, electron crystallography, electron microscopy, screening