Name: reconstitution of a kv channel into lipid membranes for structural and functional studies
Uploaded: 2013-07-13T13:00:00
Description: Watch this Scientific Journal Video about Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies at JoVE.com

The studies on KvAP in the Jiang lab have obtained significant help from Dr. Roderick MacKinnon's laboratory at the Rockefeller University. Special thanks go to Dr. Kathlynn Brown and Michael McQuire for their advice and help on our phage-screen experiments. This work was supported by grants from NIH (GM088745 and GM093271 to Q-XJ) and AHA (12IRG9400019 to Q-XJ).

1. Expression and Purification of KvAP Channel (Figure 1)
<ol>
 <li>Preparation Work - Day 0
 <ol>
 <li>Rinse the glass flasks for the bacterial culture with deionized water (diH2O) and MilliQ H2O (MQH2O) to remove trace of detergent from general dishwashing.</li>
 <li>Autoclave 1,000 ml LB medium in 2.8 L Erlenmeyer flasks (total two-liter culture as an example here). Low hardness of the water was found to be important for the successful culture of the transformed ba...

<ol>
	<li>Alberts, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biology of the Cell. , (2007).</li><li>Lee, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+lipids+and+proteins+interact+in+a+membrane:+a+molecular+approach.">How lipids and proteins interact in a membrane: a molecular approach.</a> Mol. Biosyst. 1, 203 (2005).</li><li>Lee, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+lipids+affect+the+activities+of+integral+membrane+proteins.">How lipids affect the activities of integral membrane proteins.</a> Biochim. Biophys. Acta. 1666, 62 (2004).</li><li>Anderson, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+caveolae+membrane+system.">The caveolae membrane system.</a> Annu. Rev. Biochem. 67, 199 (1998).</li><li>Simons, K., Vaz, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+systems,+lipid+rafts,+and+cell+membranes.">Model systems, lipid rafts, and cell membranes.</a> Annu. Rev. Biophys. Biomol. Struct. 33, 269 (2004).</li><li>Edidin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+lipid+rafts:+from+model+membranes+to+cells.">The state of lipid rafts: from model membranes to cells.</a> Annu. Rev. Biophys. Biomol. Struct. 32, 257 (2003).</li><li>Kapoor, R., Kim, J. H., Ingolfson, H., Andersen, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Artificial+Bilayers+for+Electrophysiology+Experiments.">Preparation of Artificial Bilayers for Electrophysiology Experiments.</a> J. Vis. Exp. (20), e1033 (2008).</li><li>Zheng, H., Liu, W., Anderson, L. Y., Jiang, Q. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid-dependent+gating+of+a+voltage-gated+potassium+channel.">Lipid-dependent gating of a voltage-gated potassium channel.</a> Nat. Commun. 2, 250 (2011).</li><li>Schmidt, D., Jiang, Q. X., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipids+and+the+origin+of+cationic+gating+charges+in+voltage+sensors.">Phospholipids and the origin of cationic gating charges in voltage sensors.</a> Nature. 444, 775 (2006).</li><li>Ruta, V., Jiang, Y., Lee, A., Chen, J., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+an+archaebacterial+voltage-dependent+K++channel.">Functional analysis of an archaebacterial voltage-dependent K+ channel.</a> Nature. 422, 180 (2003).</li><li>Jiang, Q. X., Gonen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+lipids+on+voltage-gated+ion+channels.">The influence of lipids on voltage-gated ion channels.</a> Curr. Opin. Struct. Biol. , 3408884 (2012).</li><li>Artimo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ExPASy:+SIB+bioinformatics+resource+portal.">ExPASy: SIB bioinformatics resource portal.</a> Nucleic Acids Res. 40, W597 (2012).</li><li>Cladera, J., Rigaud, J. L., Villaverde, J., Dunach, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+solubilization+and+membrane+protein+reconstitution+using+Chaps+and+Chapso.">Liposome solubilization and membrane protein reconstitution using Chaps and Chapso.</a> Eur. J. Biochem. 243, 798 (1997).</li><li>Levy, D., Bluzat, A., Seigneuret, M., Rigaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+study+of+liposome+and+proteoliposome+reconstitution+involving+Bio-Bead-mediated+Triton+X-100+removal.">A systematic study of liposome and proteoliposome reconstitution involving Bio-Bead-mediated Triton X-100 removal.</a> Biochim. Biophys. Acta. 1025, 179 (1990).</li><li>Young, H. S., Rigaud, J. L., Lacapere, J. J., Reddy, L. G., Stokes, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+tubular+crystals+by+reconstitution+of+detergent-solubilized+Ca2(+)-ATPase.">How to make tubular crystals by reconstitution of detergent-solubilized Ca2(+)-ATPase.</a> Biophys. J. 72, 2545 (1997).</li><li>Levy, D., Gulik, A., Bluzat, A., Rigaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+the+sarcoplasmic+reticulum+Ca(2+)-ATPase:+mechanisms+of+membrane+protein+insertion+into+liposomes+during+reconstitution+procedures+involving+the+use+of+detergents.">Reconstitution of the sarcoplasmic reticulum Ca(2+)-ATPase: mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents.</a> Biochim. Biophys. Acta. 1107, 283 (1992).</li><li>Cohen, F. S., Zimmerberg, J., Finkelstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+of+phospholipid+vesicles+with+planar+phospholipid+bilayer+membranes.+II.+Incorporation+of+a+vesicular+membrane+marker+into+the+planar+membrane.">Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane.</a> The Journal of General Physiology. 75, 251 (1980).</li><li>Fuks, B., Homble, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+and+electrical+properties+of+planar+lipid+membranes+from+thylakoid+lipids.">Permeability and electrical properties of planar lipid membranes from thylakoid lipids.</a> Biophysical Journal. 66, 1404 (1994).</li><li>Hanke, W., Schlue, W. -. R., Sattelle, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+Lipid+Bilayers.+Methods+and+Applications.">Planar Lipid Bilayers. Methods and Applications.</a> Biological Techniques. , 133 (1993).</li><li>Tien, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bilayer lipid membranes (BLM). Theory and Practice. , 655-65 (1974).</li><li>Pagano, R. E., Ruysschaert, J. M., Miller, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+composition+of+some+lipid+bilayer+membranes+in+aqueous+solution.">The molecular composition of some lipid bilayer membranes in aqueous solution.</a> The Journal of Membrane Biology. 10, 11 (1972).</li><li>Henn, F. A., Thompson, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+lipid+bilayer+membranes+separating+two+aqueous+phases:+composition+studies.">Properties of lipid bilayer membranes separating two aqueous phases: composition studies.</a> Journal of Molecular Biology. 31, 227 (1968).</li><li>Tao, X., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+Kv1.2+and+paddle+chimera+Kv+channels+in+planar+lipid+bilayers.">Functional analysis of Kv1.2 and paddle chimera Kv channels in planar lipid bilayers.</a> J. Mol. Biol. 382, 24 (2008).</li><li>Cohen, F. S., Akabas, M. H., Zimmerberg, J., Finkelstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+affecting+the+fusion+of+unilamellar+phospholipid+vesicles+with+planar+bilayer+membranes.">Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes.</a> The Journal of Cell Biology. 98, 1054 (1984).</li><li>Smith, G. P., Petrenko, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+Display.">Phage Display.</a> Chem. Rev. 97, 391-39 (1997).</li><li>McGuire, M. J., Li, S., Brown, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopanning+of+phage+displayed+peptide+libraries+for+the+isolation+of+cell-specific+ligands.">Biopanning of phage displayed peptide libraries for the isolation of cell-specific ligands.</a> Methods Mol. Biol. 504, 291 (2009).</li><li>Rigaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+proteins:+functional+and+structural+studies+using+reconstituted+proteoliposomes+and+2-D+crystals.">Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals.</a> Braz. J. Med. Biol. Res. 35, 753 (2002).</li><li>Chami, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+octyl+beta-thioglucopyranoside+in+two-dimensional+crystallization+of+membrane+proteins.">Use of octyl beta-thioglucopyranoside in two-dimensional crystallization of membrane proteins.</a> J. Struct. Biol. 133, 64 (2001).</li><li>Kuhlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+crystallization+of+membrane+proteins.">Two-dimensional crystallization of membrane proteins.</a> Q. Rev. Biophys. 25, 1 (1992).</li><li>Rigaud, J. L., Levy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+into+liposomes.">Reconstitution of membrane proteins into liposomes.</a> Methods Enzymol. 372, 65 (2003).</li><li>Walz, T., Grigorieff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Crystallography+of+Two-Dimensional+Crystals+of+Membrane+Proteins.">Electron Crystallography of Two-Dimensional Crystals of Membrane Proteins.</a> J. Struct. Biol. 121 (2), 142 (1998).</li><li>Signorell, G. A., Kaufmann, T. C., Kukulski, W., Engel, A., Remigy, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+2D+crystallization+of+membrane+proteins+using+methyl-beta-cyclodextrin.">Controlled 2D crystallization of membrane proteins using methyl-beta-cyclodextrin.</a> J. Struct. Biol. 157, 321 (2007).</li><li>Vink, M., Derr, K., Love, J., Stokes, D. L., Ubarretxena-Belandia, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+strategy+to+screen+2D+crystallization+trials+of+membrane+proteins.">A high-throughput strategy to screen 2D crystallization trials of membrane proteins.</a> J. Struct. Biol. 160, 295 (2007).</li><li>Iacovache, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2DX+robot:+a+membrane+protein+2D+crystallization+Swiss+Army+knife.">The 2DX robot: a membrane protein 2D crystallization Swiss Army knife.</a> J. Struct. Biol. 169, 370 (2010).</li></ol>

The reconstitution of the KvAP channels into different membranes has been used in several studies 8-10. Following the idea of ensuring the distribution of lipids between detergent/lipid mixed micelles and the protein/detergent/lipid mixed micelles, we are able to reach nearly complete reconstitution of the KvAP into membranes made of very different lipids. Each tetrameric KvAP channel needs ~100 lipid molecules to fully cover its transmembrane domain. The essential requirement is to allow enough lipid molecule...

Cells exchange materials and information with their environment through the functions of specific membrane proteins 1. Membrane proteins in cell membranes function as pumps, channels, receptors, intramembrane enzymes, linkers and structural supporters across membranes. Mutations that affect the membrane proteins have been related to many human diseases. In fact, many membrane proteins have been the primary drug targets because they are important and easily accessible in cell membranes. It is therefore very important to understand the structure and function of various membrane proteins in membranes, and make it possible to devise novel methods to alleviate the detrimental effects from the mutant proteins in human diseases.
Lipids surround all membrane proteins integrated in bilayers 2, 3. In eukaryotic membranes, the various different types of lipids are known to be organized into microdomains 4, 5. Many membrane proteins were shown to be distributed among these microdomains as well as the bulky fluid phase of membranes 3, 6. The mechanism underlying the organization of the microdomains and the delivery of membrane proteins into them and the physiological significance of such distributions are clearly important but remain poorly understood. One major technical difficulty in studying the lipid effects on membrane proteins is the reliable reconstitution of biochemically purified membrane proteins into membranes of well-controlled lipid composition so that almost all reconstituted proteins are functional 7. In the past few years, we developed methods to reconstitute the prototype voltage-gated potassium channel from A. pernix (KvAP) into various membrane systems for structural and functional studies 8-10. The data from others and us together showed that the lipids are likely a determinant in the conformational changes of the voltage-sensing domains of a voltage-gated ion channel and may shape the structures of some of these channels 11. In the next, we will provide a detailed description of our methods and will offer critical technical tips that will likely ensure the successful reproduction of our results as well as the extension of our methods to the studies of other membrane proteins.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tryptone</td>
 <td>RPI Corp.</td>
 <td>T60060</td>
 <td></td>
 </tr>
 <tr>
 <td>Yeast Extract</td>
 <td>RPI Corp.</td>
 <td>Y20020</td>
 <td></td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Fisher</td>
 <td>S271-3</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>RPI Corp.</td>
 <td>T60040</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium Chloride</td>
 <td>Fisher</td>
 <td>BP366-500</td>
 <td></td>
 </tr>
 <tr>
 <td>n-Dodecyl-&beta;-D-Maltoside</td>
 <td>Affymetrix</td>
 <td>D322S</td>
 <td>Sol-grade</td>
 </tr>
 <tr>
 <td>n-Octyl-&beta;-D-Glucoside </td>
 <td>Affymetrix</td>
 <td>O311</td>
 <td>Ana-grade</td>
 </tr>
 <tr>
 <td>Aprotinin</td>
 <td>RPI Corp.</td>
 <td>A20550-0.05</td>
 <td></td>
 </tr>
 <tr>
 <td>Leupeptin</td>
 <td>RPI Corp.</td>
 <td>L22035-0.025</td>
 <td></td>
 </tr>
 <tr>
 <td>Pepstatin A</td>
 <td>RPI Corp.</td>
 <td>P30100-0.025</td>
 <td></td>
 </tr>
 <tr>
 <td>PMSF</td>
 <td>SIGMA</td>
 <td>P7626</td>
 <td></td>
 </tr>
 <tr>
 <td>Dnase I</td>
 <td>Roche</td>
 <td>13407000</td>
 <td></td>
 </tr>
 <tr>
 <td>Bio-Bead SM-2</td>
 <td>Bio-Rad</td>
 <td>152-3920</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>RPI Corp.</td>
 <td>H75030</td>
 <td></td>
 </tr>
 <tr>
 <td>POPE</td>
 <td>Avanti Polar Lipids</td>
 <td>850757C</td>
 <td></td>
 </tr>
 <tr>
 <td>POPG</td>
 <td>Avanti Polar Lipids</td>
 <td>840457C</td>
 <td></td>
 </tr>
 <tr>
 <td>DOGS</td>
 <td>Avanti Polar Lipids</td>
 <td>870314C</td>
 <td></td>
 </tr>
 <tr>
 <td>DMPC </td>
 <td>Avanti Polar Lipids</td>
 <td>850345C</td>
 <td></td>
 </tr>
 <tr>
 <td>Biotin-DOPE </td>
 <td>Avanti Polar Lipids</td>
 <td>870282C</td>
 <td></td>
 </tr>
 <tr>
 <td>DOTAP</td>
 <td>Avanti Polar Lipids</td>
 <td>890890C</td>
 <td></td>
 </tr>
 <tr>
 <td>NeutrAvidin agarose beads </td>
 <td>Piercenet</td>
 <td>29200</td>
 <td></td>
 </tr>
 <tr>
 <td>Dialysis Tubing</td>
 <td>Spectrum Laboratories, Inc</td>
 <td>132-570</td>
 <td></td>
 </tr>
 <tr>
 <td>Pentane</td>
 <td>Fisher</td>
 <td>R399-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Decane</td>
 <td>TCI America</td>
 <td>D0011</td>
 <td></td>
 </tr>
 <tr>
 <td>MTS-PEG5000</td>
 <td>Toronto Research Cemicals</td>
 <td>M266501</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

The general flow of the experiments for purifying the KvAP channel into biochemical homogeneity is described in Figure 1A. Typical samples during the expression and purification of the protein is showed in the SDS-PAGE gel in Figure 1B. The protein after the IMAC purification is relatively pure. The yield of the KvAP channel is about 1.0 mg/Liter culture.
Solubilization of lipid vesicles with detergents needs to be worked out for each pair of lipid vs. deterge...

Watch this Scientific Journal Video about Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies at JoVE.com

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

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 ...

reconstitution-kv-channel-into-lipid-membranes-for-structural

Research

JoVE Journal

Biology

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 ...

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

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.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

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 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). 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&amp;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.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...