We are very grateful to Dr. H. Mchaourab for providing access to the EPR and DEER spectrometers and Dr. T. Rosenbaum for providing the full-length cysteine-less TRPV1 plasmid.

The authors have nothing to disclose.

Current technologies for expression and purification of mammalian membrane proteins have made it possible to obtain sufficient amounts of protein for spectroscopic studies14,15,16,42. Here, we have adapted these technologies to express, purify, reconstitute, and perform spectroscopic analyses in TRPV1.
Among the critical steps in the protocol, below are the ones that...

With recent advances in single-particle cryo-electron microscopy (cryo-EM), mammalian ion channel structures have been obtained at an extraordinary rate. Particularly, structural studies of polymodal ion channels, such as the transient receptor potential vanilloid 1 (TRPV1), have provided further understanding of its activation mechanisms1,2,3,4,5. However, dynamic information about ion channels embedded in a membrane environment is required to understand their polymodal gating and drug-binding mechanisms.
Electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) spectroscopies have provided some of the most definitive mechanistic models for ion channels6,7,8,9,10,11,12,13. These approaches have been mainly restricted to the examination of prokaryotic and archeal ion channels that yield a large amount of detergent-purified proteins when overexpressed in bacteria. With the development of eukaryotic membrane proteins production in insect and mammalian cells for functional and structural characterization14,15,16, it is now possible to obtain biochemical amounts of detergent-purified proteins for spectroscopic studies.
The EPR and DEER signals arise from a&#160;paramagneticspin label (SL) (i.e., methanethiosulfonate) attached to a single-cysteine residue in the protein. The spin-labels report three types of structural information: motion, accessibilities, and distances. This information allows determining whether residues are buried within the protein or are exposed to the membrane or aqueous environment in the apo and ligand-bound states13,17,18,19. In the context of a high-resolution structure (when available), the EPR and DEER data provide a collection of constraints for deriving dynamic models in their native environment while monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Moreover, flexible regions that might be difficult to determine by X-ray crystallography or cryo-EM could be obtained by using these environmental data sets to assign secondary structures as well as location within the protein20. Cryo-EM structures obtained in lipid nanodiscs provided valuable information about the gating of ion channels3,21,22,23,24,25; however, spectroscopic approaches could provide dynamic information from conformational states (e.g., thermal changes) that might be difficult to determine using cryo-EM.
Many difficulties must be overcome to implement EPR and DEER, including lack of protein function when removing all cysteine residues (especially abundant in mammalian channels), low protein yield, protein instability during purification and after spin labeling, and protein aggregation in detergent or liposomes. Here, we have designed protocols to overcome these critical barriers and have obtained DEER and EPR spectra information for a mammalian sensory receptor. The purpose here is to describe methodologies for the expression, purification, labeling, and reconstitution of a functional minimal cysteine-less rat TRPV1 (eTRPV1) construct for spectroscopic analyses. This methodology is appropriate for those membrane proteins that keep their function despite the removal of cysteine residues or that contain cysteine forming disulfide-bonds. This collection of protocols could be adapted for the spectroscopic analysis of other mammalian ion channels.

<table><tbody><tr><td>QuikChange Lightning Site-Directed Mutagenesis Kit</td><td>Agilent Technologies</td><td>210519-5</td><td></td></tr><tr><td>2-Propanol (Isopropanol)</td><td>Fisher Scientific</td><td>A416</td><td></td></tr><tr><td>Albumin Bovine Serum (BSA)</td><td>GoldBio.com</td><td>A-420-10</td><td></td></tr><tr><td>Amylose resin</td><td>NEB</td><td>E8021L</td><td></td></tr><tr><td>Aprotinin</td><td>GoldBio.com</td><td>A-655-25</td><td></td></tr><tr><td>Asolectin from Soybean</td><td>Sigma</td><td>11145</td><td></td></tr><tr><td>Bac-to-Bac Baculovirus Expression System</td><td>Invitrogen Life Technologies</td><td>10359016</td><td></td></tr><tr><td>Biobeads SM-2 Adsorbents&amp;nbsp;</td><td>Bio-Rad</td><td>152-3920</td><td></td></tr><tr><td>Borosilicate glass pipettes (3.5&#039;&#039;) (oocyte inyection)</td><td>Drummond Scientific</td><td>3-000-203 G/X</td><td></td></tr><tr><td>Borosilicate glass pipettes (oocyte recordings)</td><td>Sutter Instrument</td><td>B150-110-10HP</td><td></td></tr><tr><td>CaCl2 2H2O</td><td>Fisher Scientific</td><td>C79</td><td></td></tr><tr><td>Carbenicillin (Disodium)</td><td>GoldBio.com</td><td>C-103-5</td><td></td></tr><tr><td>Cellfectin Reagent</td><td>Invitrogen Life Technologies</td><td>10362-010</td><td></td></tr><tr><td>cellSens</td><td>Olympus</td><td></td><td></td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>C606SK</td><td></td></tr><tr><td>Collagenase Type 1</td><td>Worthington-Biochem</td><td>LS004196</td><td></td></tr><tr><td>Critiseal</td><td>VWR</td><td>18000-299</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma&amp;nbsp;</td><td>G8270</td><td></td></tr><tr><td>D-(+)-Maltose Monohydrate</td><td>Fisher Scientific</td><td>BP684</td><td></td></tr><tr><td>DDM (n-Docecyl-B-D-Maltopyranoside)</td><td>Anatrace</td><td>D310S</td><td></td></tr><tr><td>High glucose medium (Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium)</td><td>Sigma</td><td>D0572&amp;nbsp;</td><td></td></tr><tr><td>Disposable PD-10 Desalting Columns</td><td>GE Healthcare</td><td>45-000-148</td><td></td></tr><tr><td>EGTA</td><td>Fisher Scientific</td><td>O2783</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Invitrogen Life Technologies</td><td>10082-147</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Life Technologies</td><td>F-14201</td><td></td></tr><tr><td>GenCatch Plus Plasmid DNA Mini-Prep Kit</td><td>Epoch Life Science, Inc</td><td>2160250</td><td></td></tr><tr><td>GenCatch PCR Cleanup Kit</td><td>Epoch Life Science, Inc</td><td>2360050</td><td></td></tr><tr><td>Gentamicin Sulfate</td><td>Lonza</td><td>17-518Z</td><td></td></tr><tr><td>Glass capillary (25 &amp;micro;l)</td><td>VWR</td><td>53432-761</td><td></td></tr><tr><td>Glass Flask 2800 mL</td><td>Pyrex USA</td><td>4423-2XL</td><td></td></tr><tr><td>Glycerol</td><td>Fisher BioReagents</td><td>BP229</td><td></td></tr><tr><td>HEK293S GnTl-</td><td>ATCC</td><td>CRL-3022</td><td></td></tr><tr><td>HEPES</td><td>Sigma&amp;nbsp;</td><td>H4034</td><td></td></tr><tr><td>IPTG (isopropyl-thio-B-galactoside)</td><td>GoldBio.com</td><td>I2481C25</td><td></td></tr><tr><td>Kanamycin Sulfate</td><td>Fisher Scientific</td><td>BP906-5</td><td></td></tr><tr><td>KCl</td><td>Fisher Chemical</td><td>P217</td><td></td></tr><tr><td>LB Broth, Miller</td><td>Fisher bioReagents</td><td>BP1426</td><td></td></tr><tr><td>Leupeptin Hemisulfate</td><td>GoldBio.com</td><td>L-010-5</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen Life Technologies</td><td>11668-019</td><td></td></tr><tr><td>MgCl2 6H2O</td><td>Fisher Scientific</td><td>BP214</td><td></td></tr><tr><td>MgSO4 7H2O</td><td>Fisher Scientific</td><td>BP213</td><td></td></tr><tr><td>mMESSAGE mMACHINE T7 Kit</td><td>Ambion</td><td>AM1344</td><td></td></tr><tr><td>MOPS</td><td>Fisher bioReagents</td><td>BP2936</td><td></td></tr><tr><td>MTSL (1-Oxyl-2,2,5,5-tetramethylpyrrolidin-3-yl) Methyl Methanethiosulfonate</td><td>Toronto Research Chemicals, Inc</td><td>O873900</td><td></td></tr><tr><td>NaCl</td><td>Fisher Chemical</td><td>S271</td><td></td></tr><tr><td>Opti-MEM</td><td>Life Technologies</td><td>31985-062</td><td></td></tr><tr><td>Pepstatin A</td><td>GoldBio.com</td><td>P-020-5</td><td></td></tr><tr><td>Pluronic Acid F-127 (20%)</td><td>PromoKine&amp;nbsp;&amp;nbsp;</td><td>CA707-59004</td><td></td></tr><tr><td>PMSF</td><td>GoldBio.com</td><td>P4170</td><td></td></tr><tr><td>Poly-L-lysine Solution</td><td>Sigma-Aldrich</td><td>P4707</td><td></td></tr><tr><td>Rneasy Mini Kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Sealed capillary</td><td>VitroCom</td><td>special order</td><td></td></tr><tr><td>SF-900 II SFM (insect cell medium)</td><td>Gibco, Life Technologies</td><td>10902-088</td><td></td></tr><tr><td>Sf9 Cells (SFM Adapted)</td><td>Invitrogen Life Technologies</td><td>11496-015</td><td></td></tr><tr><td>Soybean Polar Lipid Extract</td><td>Avanti Polar Lipids, Inc</td><td>541602C</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S25590</td><td></td></tr><tr><td>Superose 6 Increase 10/300 GL</td><td>GE Healthcare</td><td>29091596</td><td></td></tr><tr><td>TCEP HCl</td><td>GoldBio.com</td><td>TCEP1</td><td></td></tr><tr><td>Tetracyclin Hydrochloride</td><td>Fisher Scientific</td><td>BP912-100</td><td></td></tr><tr><td>Tris Base</td><td>Fisher BioReagents</td><td>BP152</td><td></td></tr><tr><td>Tryptone</td><td>Difco</td><td>0123-01</td><td></td></tr><tr><td>X-gal</td><td>GoldBio.com</td><td>X4281C</td><td></td></tr><tr><td>Xenopus oocytes</td><td>Nasco</td><td>LM00935M</td><td></td></tr><tr><td>XL1 - Blue Competent Cells</td><td>Agilent Technologies, Inc</td><td>200249</td><td></td></tr><tr><td>Yeast Extract</td><td>Difco</td><td>0127-01-7</td><td></td></tr><tr><td>Econo-Pack chromatography column</td><td>Bio-Rad</td><td>7321010</td><td></td></tr><tr><td>Mini-PROTEAN TGX Stain-Free Precast Gels</td><td>Bio-Rad</td><td>17000436</td><td></td></tr><tr><td>pFastBac1 Expression Vector</td><td>Invitrogen Life Technologies</td><td>10360-014</td><td></td></tr><tr><td>DH10Bac Competent Cells</td><td>Invitrogen Life Technologies</td><td>10361-012</td><td></td></tr><tr><td>Critiseal capillary tube sealant</td><td>Leica Microsystems</td><td>02-676-20</td><td></td></tr><tr><td>ABI Model 3130XL Genetic Analyzers</td><td>Applied Biosystems</td><td>4359571</td><td></td></tr><tr><td>Transfer pipete</td><td>Fishebrand</td><td>13-711-9AM</td><td></td></tr><tr><td>Nanoject II</td><td>Drummond Scientific</td><td>3-000-204</td><td></td></tr></tbody></table>

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.

Functional Characterization of the Minimal Cysteine-less TRPV1 Construct (eTRPV1) and Single-cysteine Mutants
The first step toward spectroscopic studies is to engineer and characterize cysteine-less protein constructs (Figure 2A) that are functional and yield biochemical amounts of proteins. eTRPV1 is functional as determined by Ca2+ imaging and TEVC (Figure 2B...

Watch this Scientific Journal Video about Purification and Reconstitution of TRPV1 for Spectroscopic Analysis at JoVE.com

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

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 ...

purification-and-reconstitution-of-trpv1-for-spectroscopic-analysis

Research

JoVE Journal

Biochemistry

7.9K Views.  University of Tennessee Health Science Center. Dit artikel wordt beschreven specifieke methoden voor biochemische hoeveelheid wasmiddel-ontbindend TRPV1 spectroscopische p.a.. De gecombineerde protocollen bieden biochemische en biofysische hulpmiddelen die kunnen worden aangepast om de structurele en functionele studies voor zoogdieren ionenkanalen in een membraan-gecontroleerde omgeving.

Video: Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Genetics

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102-103 repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 &#181;g, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.

Key steps of protein function, in particular backbone conformational changes and proton transfer reactions, often take place in the microsecond to millisecond time scale. These dynamical processes can be studied by time-resolved step-scan Fourier-transform infrared spectroscopy, in particular for proteins whose function is triggered by light.

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards ...

Engineering

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

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.

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 ...

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

Biology

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Bioengineering

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, ...

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step toward a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented.

This protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided alongside directions to prevent potential pitfalls.

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take ...

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 ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

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