Name: expression and purification of the human lipid-sensitive cation channel trpc3 for structural determination by single-particle cryo-electron microscopy
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy at JoVE

We thank G. Zhao and X. Meng for the support with data collection at the David Van Andel Advanced Cryo-Electron Microscopy Suite. We appreciate the VARI High-Performance Computing team for computational support. We give our gratitude to N. Clemente, D. Dues, J. Floramo, Y. Huang, Y. Kim, C. Mueller, B. Roth, and Z. Ruan for comments that greatly improved this manuscript. We thank D. Nadziejka for editorial support for this manuscript. This work was supported by internal VARI funding.

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The authors have nothing to disclose.

Structural determination of proteins by cryo-EM has revolutionized the field of structural biology in the past few years, thanks to the development of new cameras and algorithms that significantly speeds up the structure determination of proteins that do not readily crystalize, particularly membrane proteins. Despite all of the recent advances in the cryo-EM technique, the preparation of purified proteins sufficient in quality and quantity to facilitate high-quality imaging often remains time-consuming, costly, and challenging. The ability to rapidly express and screen multiple gene constructs under a variety of purification conditions, as described in the protocol above, improves the efficiency of this process by allowing the rapid and cost-effective production of high-quality purified protein for use in cryo-EM studies.
While the method described here provides a way to purify large quantities of mammalian membrane proteins at less cost than by direct transfection and more quickly than by generation of a stably transfected cell line, it has many steps, each of which must be optimized to provide a high-quality protein yield. Within the biochemical techniques used to produce and purify hTRPC3, there are a number of critical steps and checkpoints. The first critical checkpoint is the production of bacmid DNA. As described in the results, ideal colony selection is the first step in generation of a quality virus for protein expression. Additionally, if the concentration of bacmid DNA purified from the selected colony is less than 1 &#956;g/&#956;L, the resulting virus will not be sufficient for robust protein expression. The second critical checkpoint is the production of P1 and P2 baculovirus. Virus titer can be estimated by GFP fluorescence, as shown in Figure 1C or can be measured as described by Coleman et al.35. In addition to viral titer, a time course of infection time should be undertaken for each new construct to determine the optimal time of infection for maximal protein expression. Critical checkpoint three is the solubility and stability screening shown in Figure 2. Detergents with a high CMC, such as DDM, may provide high solubility, but they are not ideal for cryo-EM imaging because they can result in an abundance of contaminating micelles. A milder detergent, such as digitonin, can provide sufficient solubility while preserving the protein stability. The screening of additives, such as EDTA in the case of hTRPC3, is important for discovering a purification condition that results in intact and homogenous protein, probably by removing the calcium from hTRPC3, which is permeable to calcium. Critical checkpoint four is the verification of specific and efficient protein capture during the affinity column purification and the observation of an ideal protein peak during SEC-fractionation (Figure 4). Avoidance of the inclusion of contaminating protein particles while retaining all of the target protein is crucial to obtaining a high enough yield of protein for cryo-EM imaging. Alteration of the batch-binding time and ratio of resin to solubilized protein can help improve protein retention by the resin column. The overall quality of the SEC-fractionation peak is in our experience the best indicator, prior to imaging, of the quality of purified protein particles. A low or a broad peak during SEC-fractionation indicates possible problems of too few protein particles per image or the presence of contaminating protein aggregation/degradation products, respectively. Additionally, changes to the affinity tag within the construct itself has proven necessary in some cases to ensure sufficient affinity-tag binding. The final checkpoint prior to collection of cryo-EM data sets is the verification of protein particle quality by negative-stain EM. While not strictly necessary, we find that this provides an excellent opportunity to spot and correct protein quality issues before investing in the time- and cost-intensive process of collecting data by cryo-EM.
One alternative method of producing protein for cryo-EM structural studies is the direct purification of protein from the native tissue source. While this method often encounters issues with protein yield, it is an excellent alternative for proteins that are highly abundant in cells or that exist as part of a large multi-protein complex, which can be difficult to produce using a recombinant overexpression system36. However, for single proteins expressed in moderate or low amounts under physiologic conditions, the expression and purification system presented here is an efficient, relatively low-cost and high-yield method for producing purified mammalian membrane protein for cryo-EM structural studies. One method that could strongly complement that presented here is the reconstitution of purified protein into lipid nanodiscs prior to cryo-EM data collection37. Under this method, the proteins are imbedded into a small disk of lipid bilayer, probably providing a native-like microenvironment in comparison to detergent micelles, although there is no evidence so far that the structures dissolved in detergent and nanodisc are different38,39,40. Another approach is to extract the protein directly using amphipathic polymers like styrene maleic anhydride (SMA), which has been successfully used for determination of membrane protein structures41,42.
These approaches described in this manuscript have proven to be readily adaptable in our lab to a variety of other mammalian membrane proteins beyond ion channels. Therefore, we believe this protocol will be a valuable tool in the structural and functional analyses that underlie high-specificity targeted drug design. This will be particularly useful in neurodegenerative disease, cardiovascular disease, and cancer, in which ion channels are difficult but high-potential drug targets.

Calcium is involved in most cellular processes including signaling cascades, transcription control, neurotransmitter release, and hormone molecule synthesis1,2,3. The homeostatic maintenance of cytosolic free&#160;calcium is crucial to the health and function of cells. One of the major mechanisms of intracellular calcium homeostasis is store-operated calcium entry (SOCE), a process in which depletion of calcium stored in the endoplasmic reticulum (ER) triggers the opening of ion channels on the plasma membrane to facilitate the replenishment of ER calcium, which can then be used in further signaling4,5,6. Transient receptor potential channels (TRPCs), which are calcium-permeable channels belonging to the TRP superfamily, have been identified as a major participant in SOCE7,8,9 .
Among the seven members in the TRPC family, TRPC3, TRPC6, and TRPC7 form a homologue subgroup, and they are unique in the ability to be activated by the lipid secondary messenger diacylglycerol (DAG), a degradation product of the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2)10,11. TRPC3 is highly expressed in smooth muscle and in the cerebral and cerebellar regions of the brain, where it plays essential roles in calcium signaling that impact&#160;neurotransmission and neurogenesis12,13. Dysfunction of TRPC3 has been linked to central nervous system disorders, cardiovascular disorders, and certain cancers such as ovarian adenocarcinoma14,15,16. Therefore, TRPC3 holds promise as a pharmaceutical target for treatment of these diseases. The development of specifically targeted drugs acting on TRPC3 has been limited by a lack of understanding of its molecular activation mechanisms, including lipid binding sites17,18. We have reported the first atomic-resolution structure of the human TRPC3 channel (hTRPC3) and its two lipid binding sites in a closed state, providing important insights into these mechanisms19.
The key factor for determining the structure of a membrane protein at high resolution is to obtain protein of high quality. The corresponding screening of expression and purification conditions necessary to obtain high quality protein can be a time-consuming and costly endeavor. Here we present a protocol describing in detail how we identify the optimal conditions for the expression and purification of hTRPC3, which behaved poorly in our initial screening. We present several key points on how to troubleshoot and optimize the protein behavior, which lay a solid foundation for our cryo-electron microscopy (cryo-EM) studies. We use a modified baculoviral generating vector (pEG), developed by Gouaux and colleagues, which is optimized for screening assays and efficient generation of baculovirus in mammalian cells20. This expression method is appropriate for rapid and cost-effective overexpression of proteins in the mammalian cell membrane. We combine the use of this vector with a fluorescence-detection size-exclusion chromatography-based (FSEC) prescreening method21. This method uses a green fluorescent protein (GFP) tag fused to the construct of interest and improves visualization of the target protein in small, whole-cell solubilized samples. This allows for screening of protein stability in the presence of different detergents and additives, with thermostabilizing mutations, and allows the use of a small number of cells from small-scale transient transfection. In this way, a multitude of conditions can be rapidly screened before moving to a large-scale protein purification. Following expression, screening, and purification, we present a protocol for obtaining and processing images from cryo-EM to generate a de novo structural determination of the protein. We believe that the approaches described here will serve as a generalizable protocol for structural studies of TRP channel receptors and other membrane proteins.

<table><tbody><tr><td>pEG BacMam vector (pFastBacI)</td><td>addgene</td><td>31488</td><td></td></tr><tr><td>DH10&amp;alpha; cells</td><td>Life Technologies</td><td>10361-012</td><td></td></tr><tr><td>S.O.C. media</td><td>Corning</td><td>46003CR</td><td>for transformation of DH10&amp;alpha; cells for Bacmid</td></tr><tr><td>Bacmam culture plates</td><td>Teknova</td><td>L5919</td><td>for culture of transformed DH10&amp;alpha; cells</td></tr><tr><td>Incubation shaker for bacterial cells</td><td>Infors HT</td><td>Multitron standard</td><td></td></tr><tr><td>Incubated orbital shaker for insect cells</td><td>Thermo-Fisher</td><td>SHKE8000</td><td></td></tr><tr><td>Reach-in CO&lt;sub&gt;2 &lt;/sub&gt;incubator for mammalian cells</td><td>Thermo-Fisher</td><td>3951</td><td></td></tr><tr><td>Table-top orbital shaker</td><td>Thermo-Fisher</td><td>SHKE416HP</td><td>used in Reach-in CO2 incubator for mammalian cells</td></tr><tr><td>Incubator</td><td>VWR</td><td>1535</td><td>for bacterial plates</td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27106</td><td>for plasmid extraction and purification</td></tr><tr><td>Phenol:Chloroform:Isoamyl alcohol</td><td>Invitrogen</td><td>15593031</td><td>for DNA extraction</td></tr><tr><td>Sf9 cells</td><td>Life Technologies</td><td>12659017</td><td>insect cells for producing virus</td></tr><tr><td>Sf-900 media</td><td>Gibco</td><td>12658-027</td><td>insect cell media</td></tr><tr><td>FBS</td><td>Atlanta Biologicals</td><td>S11550</td><td></td></tr><tr><td>Cellfectin II</td><td>Gibco</td><td>10362100</td><td>for transfecting insect cells</td></tr><tr><td>lipofectamine 2000</td><td>Invitrogen</td><td>11668-027</td><td>for transfecting mamalian cells</td></tr><tr><td>0.2 mm syringe filter</td><td>VWR</td><td>28145-501</td><td>for filtering P1 virus</td></tr><tr><td>0.2 mm filter flasks 500ml resevoir</td><td>Corning</td><td>430758</td><td>for filtering P2 virus</td></tr><tr><td>erlenmeyer culture flask (flat bottom 2L)</td><td>Gene Mate</td><td>F-5909-2000</td><td>for culturing insect cells</td></tr><tr><td>erlenmeyer culture flask (baffled 2L)</td><td>Gene Mate</td><td>F-5909-2000B</td><td>for culturing mammalian cells</td></tr><tr><td>nanodrop 2000 spectrophotometer</td><td>Thermo-Fisher</td><td>ND-2000</td><td>for determining DNA and protein concentrations</td></tr><tr><td>HEK293</td><td>ATCC</td><td>CRL-3022</td><td>mammalian cells for producing protein</td></tr><tr><td>Freestyle 293 expression Medium</td><td>Gibco</td><td>1238-018</td><td>mammalian cell media for protein expression</td></tr><tr><td>Butyric Acid Sodium Salt</td><td>Acros</td><td>263195000</td><td>to amplify protein expression</td></tr><tr><td>PMSF</td><td>Acros</td><td>215740500</td><td>protease inhibitor</td></tr><tr><td>Aprotinin from bovine lung</td><td>Sigma-Aldrich</td><td>A1153-100MG</td><td>protease inhibitor</td></tr><tr><td>Leupeptin hydrochloride</td><td>Sigma-Aldrich</td><td>24125-16-4</td><td>protease inhibitor</td></tr><tr><td>pepstatin A</td><td>Fisher Scientific</td><td>BP2671-250</td><td>protease inhibitor</td></tr><tr><td>digitonin</td><td>EMD Millipore</td><td>300410</td><td>detergent - to solubilize protein from membrane</td></tr><tr><td>imidazole</td><td>Sigma</td><td>792527</td><td>to elute protein from resin column</td></tr><tr><td>TALON resin</td><td>Clonetech</td><td>635504</td><td>for affinity purification by His-tag</td></tr><tr><td>superose6 incease columns</td><td>GE</td><td>29091596; 29091597</td><td>for HPLC and FPLC</td></tr><tr><td>Prominence Modular HPLC System</td><td>Shimadzu</td><td>See Below</td><td></td></tr><tr><td>Controller Module</td><td>&quot;</td><td>CBM20A</td><td></td></tr><tr><td>Solvent Delivery System</td><td>&quot;</td><td>LC30AD</td><td></td></tr><tr><td>Fluorescence Detector</td><td>&quot;</td><td>RF20AXS</td><td></td></tr><tr><td>Autosampler with Cooling</td><td>&quot;</td><td>SIL20ACHT</td><td></td></tr><tr><td>Pure FPLC System with Fractionator</td><td>Akta</td><td></td><td></td></tr><tr><td>thrombin (alpha)</td><td>Haematologic Technologies Incorporated</td><td>HCT-0020 Human alpha</td><td>for cleaving GFP tag</td></tr><tr><td>Amicon Ultra 15 mL 100K centrifugal filter tube</td><td>Millipore</td><td>UFC910008</td><td>for concentrating protein</td></tr><tr><td>EDTA</td><td>Fisher</td><td>E478500</td><td>for stabilizing protein</td></tr><tr><td>400 mesh carbon-coated copper grids</td><td>Ted Pella Inc.</td><td>01754-F</td><td>grids for negative stain</td></tr><tr><td>Quantifoil holey carbon grid (gold, 1.2/1.3&amp;thinsp;&amp;mu;m size/hole space, 300 mesh)</td><td>Electron Microscopy Sciences</td><td>Q3100AR1.3</td><td>grids for Cryo-EM</td></tr><tr><td>Vitrobot Mark III</td><td>FEI</td><td></td><td>for preparing sample grids by liquid ethane freezing</td></tr><tr><td>liquid nitrogen</td><td>Dura-Cyl</td><td>UN1977</td><td></td></tr><tr><td>ethane gas</td><td>Airgas</td><td>UN1035</td><td></td></tr><tr><td>Solarus Plasma System</td><td>Gatan</td><td>Model 950</td><td>for cleaning grids before sample freezing</td></tr><tr><td>Tecnai Spirit electron microscope</td><td>FEI</td><td></td><td>for negative stain EM imaging</td></tr><tr><td>Talos Arctica electron microsocope</td><td>FEI</td><td></td><td>for screening and low resolution imaging of Cryo-EM grids</td></tr><tr><td>Titan Krios electron microscope</td><td>FEI</td><td></td><td>for high-resolution Cryo-EM imaging</td></tr><tr><td>Software</td><td></td><td></td><td></td></tr><tr><td>Gautomatch software</td><td>http://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/</td><td></td><td>to pick particles from micrographs</td></tr><tr><td>Relion 2.1 software</td><td>https://github.com/3dem/relion</td><td></td><td>to construct 2D and 3D classification</td></tr><tr><td>CryoSPARC software</td><td>https://cryosparc.com/</td><td></td><td>to generate an initial structure model</td></tr><tr><td>Frealign software</td><td>http://grigoriefflab.janelia.org/frealign</td><td></td><td>to refine particles</td></tr><tr><td>Coot software</td><td>https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/</td><td></td><td>to build a model</td></tr><tr><td>MolProbity software</td><td>http://molprobity.biochem.duke.edu/</td><td></td><td>to evaluate the geometries of the atomic model</td></tr><tr><td>SerialEM software</td><td>http://bio3d.colorado.edu/SerialEM/</td><td></td><td>for automated serial image stack acquisition</td></tr><tr><td>MortionCor2 software</td><td>http://msg.ucsf.edu/em/software/motioncor2.html</td><td></td><td>for motion correction of summed movie stacks</td></tr><tr><td>GCTF software</td><td>https://www.mrc-lmb.cam.ac.uk/kzhang/Gctf/</td><td></td><td>for measuring defocus values in movie stacks</td></tr><tr><td>Phenix.real_space_refine software</td><td>https://www.phenix-online.org/documentation/reference/real_space_refine.html</td><td></td><td>for real space refinement of the initial 3D model</td></tr></tbody></table>

expression and purification of the human lipid-sensitive cation channel trpc3 for structural determination by single-particle cryo-electron microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

Watch this Scientific Journal Video about Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy at JoVE

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

expression-purification-human-lipid-sensitive-cation-channel-trpc3

Nanyang Technological University

Research

JoVE Journal

Biochemistry

9.4K Views.  Van Andel Institute. This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Video: Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

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&#8208;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&#8208;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.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

Environment

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Structure determination of macromolecular complexes using cryoEM has become routine for certain classes of proteins and complexes. Here, this pipeline is summarized (sample preparation, screening, data acquisition and processing) and readers are directed towards further detailed resources and variables that may be altered in the case of more challenging specimens.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

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.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Biology

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...

A Pipeline to Investigate the Structures and Signaling Pathways of Sphingosine 1-Phosphate Receptors

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of sphingolipids in the cell membrane, is one of the best-characterized LPLs that regulates a variety of cellular physiological responses via signaling pathways mediated by sphingosine 1-phosphate receptors (S1PRs). This implicated that the S1P-S1PRs signaling system is a remarkable potential therapeutic target for disorders, including multiple sclerosis (MS), autoimmune disorders, cancer, inflammation, and even COVID-19. S1PRs, a small subset of the class A G-protein coupled receptor (GPCR) family, are composed of five subtypes: S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5. The lack of detailed structural information, however, impedes the drug discovery targeting S1PRs. Here, we applied the cryo-electron microscopy method to solve the structure of the S1P-S1PRs complex, and elucidated the mechanism of activation, selective drug recognition, and G-protein coupling by using cell-based functional assays. Other lysophospholipid receptors (LPLRs) and GPCRs can also be studied using this strategy.

S1P exerts its diverse physiological effects through the S1P receptors (S1PRs) subfamily. Here, a pipeline is described to expound on the structures and function of S1PRs.

Lysophospholipids (LPLs) are bioactive lipids that include sphingosine 1-phosphate (S1P), lysophosphatidic acid, etc. S1P, a metabolic product of ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

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.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Summary

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Chapters in this video

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Expression and Purification of Mammalian Bestrophin Ion Channels

Single Particle Cryo-Electron Microscopy: From Sample to Structure

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

Purification of Endogenous Drosophila Transient Receptor Potential Channels

A Pipeline to Investigate the Structures and Signaling Pathways of Sphingosine 1-Phosphate Receptors

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae