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E., Friedrich, T., Bamberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta+subunit+of+the+Na+/K+-ATPase+follows+the+conformational+state+of+the+holoenzyme.">The beta subunit of the Na+/K+-ATPase follows the conformational state of the holoenzyme.</a> J. Gen. Physiol. 125, 505-520 (2005).</li><li>Miller, A. J., Zhou, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+oocytes+as+an+expression+system+for+plant+transporters.">Xenopus oocytes as an expression system for plant transporters.</a> BBA - Biomembranes. 1465, 343-358 (2000).</li><li>Cha, A., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+Voltage-Dependent+Conformational+Changes+in+the+Shaker+K++Channel+with+Fluorescence.">Characterizing Voltage-Dependent Conformational Changes in the Shaker K+ Channel with Fluorescence.</a> Neuron. 19, 1127-1140 (1997).</li><li>Mannuzzu, L. M., Moronne, M. M., Isacoff, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Physical+Measure+of+Conformational+Rearrangement+Underlying+Potassium+Channel+Gating.">Direct Physical Measure of Conformational Rearrangement Underlying Potassium Channel Gating.</a> Science. 271, 213-216 (1996).</li><li>Koch, H. P., Larsson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-scale+molecular+motions+accomplish+glutamate+uptake+in+human+glutamate+transporters.">Small-scale molecular motions accomplish glutamate uptake in human glutamate transporters.</a> J. Neurosci. 25, 1730-1736 (2005).</li><li>Dempski, R. E., Hartung, K., Friedrich, T., Bamberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorometric+Measurements+of+Intermolecular+Distances+between+the+&#945;-+and+&#946;-Subunits+of+the+Na+/K+-ATPase.">Fluorometric Measurements of Intermolecular Distances between the &#945;- and &#946;-Subunits of the Na+/K+-ATPase.</a> J. Biol. Chem. 281, 36338-36338 (2006).</li><li>Geys, S. A., Bamberg, E., Dempski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-Dependent+Effects+on+the+Conformational+Equilibrium+of+the+Na+,K+-ATPase+As+Monitored+by+Voltage+Clamp+Fluorometry.">Ligand-Dependent Effects on the Conformational Equilibrium of the Na+,K+-ATPase As Monitored by Voltage Clamp Fluorometry.</a> Biophys. J. 96, 4561-4561 (2009).</li><li>Geibel, S., Kaplan, J. H., Bamberg, E., Friedrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Dynamics+of+the+Na+/K+-ATPase+Probed+by+Voltage+Clamp+Fluorometry.">Conformational Dynamics of the Na+/K+-ATPase Probed by Voltage Clamp Fluorometry.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 964-969 (2003).</li><li>Lorenz, L., Pusch, M., Jentsch, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heteromultimeric+CLC+chloride+channels+with+novel+properties.">Heteromultimeric CLC chloride channels with novel properties.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 13362-13366 (1996).</li><li>Mruk, K., Kobertz, W. 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Physiol. 112, 391-391 (1998).</li><li>F&#246;rster, 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> Ann Physik. 2, 55-75 (1948).</li><li>Dong, X., Stothard, P., Forsythe, I. J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PlasMapper:+a+web+server+for+drawing+and+auto-annotating+plasmid+maps.">PlasMapper: a web server for drawing and auto-annotating plasmid maps.</a> Nucleic Acids Res. 32, W660-W664 (2004).</li></ol>

No conflicts of interest declared.

The experimental approach that we describe combines site-specific fluorophore labeling and two electrode voltage clamp to investigate the relationship between structure and function of membrane proteins. This technique can be used to obtain time-resolved information on the conformational dynamics of membrane proteins during ion transport. Furthermore, this approach can be tailored to work with various proteins such as ion pumps, ion channels and transporters. 
In addition to investigating the conformational dynamics at a specific residue, it is also possible to use fluorescence resonance energy transfer to determine distance constraints within a holoenzyme. The determination of distances between residues as well as measuring the relative movement between subunits can help solve key questions involving gating mechanisms.

<table border="1">
	<tbody>
		<tr>
			<th>Reagent/Equipment</th>
			<th>Company</th>
			<th>Reagent/Equipment</th>
			<th>Company</th>
		</tr>
		<tr>
			<td>100W Tungsten Light Source</td>
			<td>Carl Zeiss Microimaging</td>
			<td>fluorescein-5-maleimide</td>
			<td>Invitrogen</td>
		</tr>
		<tr>
			<td>1B150F-4</td>
			<td>World Precision Instruments</td>
			<td>High-Pure PCR Extraction Kit</td>
			<td>Roche</td>
		</tr>
		<tr>
			<td>3.0-4.0 Ethicon Vicryl</td>
			<td>Johnson &amp; Johnson</td>
			<td>mMessage mMachine Kit</td>
			<td>Ambion</td>
		</tr>
		<tr>
			<td>475AF40 excitation filter</td>
			<td>Omega Optical</td>
			<td>MS-222</td>
			<td>Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>505DRLP dichroic mirror</td>
			<td>Omega Optical</td>
			<td>Nucleospin Plasmid Kit</td>
			<td>Macherey-Nagel</td>
		</tr>
		<tr>
			<td>Macherey-Nagel</td>
			<td>Omega Optical</td>
			<td>Nanodrop 2000c Sprectrophotometer</td>
			<td>Thermo Scientific</td>
		</tr>
		<tr>
			<td>535DF50 excitation filter</td>
			<td>Omega Optical</td>
			<td>PC-10 Micropipette Puller</td>
			<td>Narishige</td>
		</tr>
		<tr>
			<td>560DRLP dichroic mirror</td>
			<td>Omega Optical</td>
			<td>pCLAMP 10 Software</td>
			<td>Axon Instruments</td>
		</tr>
		<tr>
			<td>565ALP emission filter</td>
			<td>Omega Optical</td>
			<td>PfuTurbo DNA Polymerase</td>
			<td>Strategene</td>
		</tr>
		<tr>
			<td>565EFLP emission filter</td>
			<td>Omega Optical</td>
			<td>PIN-022A Photodiode</td>
			<td>United Detector Technologies</td>
		</tr>
		<tr>
			<td>570DRLP dichroic mirror</td>
			<td>Omega Optical</td>
			<td>Polarized Filters</td>
			<td>Linos Phtonics Inc.</td>
		</tr>
		<tr>
			<td>Linos Phtonics Inc.</td>
			<td>Omega Optical</td>
			<td>QuickChange Site-Directed Mutagenisis Kit</td>
			<td>Stratagene</td>
		</tr>
		<tr>
			<td>Axio Examiner Fluorescence Microscope</td>
			<td>Carl Zeiss MicroImaging GMBH</td>
			<td>RC-10 Fluorescence Chamber</td>
			<td>Warner Instruments</td>
		</tr>
		<tr>
			<td>Warner Instruments</td>
			<td>Life Technologies</td>
			<td>tetramethylrhodamine-6-maleimide</td>
			<td>Invitrogen</td>
		</tr>
		<tr>
			<td>Digidata 1440A data acquisition system</td>
			<td>Axon Instruments</td>
			<td>TOP10 Electrocompetent Cells</td>
			<td>Invitrogen</td>
		</tr>
		<tr>
			<td>Dpn I</td>
			<td>New England Biolabs</td>
			<td>Turbo Tec-05X amplifier</td>
			<td>npi</td>
		</tr>
	</tbody>
</table>

examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Watch this Scientific Journal Video about Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling at JoVE.com

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

examining-conformational-dynamics-membrane-proteins-situ-with-site

Research

JoVE Journal

Biology

14.5K visualizações.  Worcester Polytechnic Institute. Vamos descrever um método que mede a cinética de transporte de íons de proteínas da membrana juntamente com site-specific análise das mudanças conformacionais usando fluorescência em células individuais. Esta técnica é adaptável a canais iônicos, transportadores e bombas de íons e pode ser utilizado para determinar restrições de distância entre as subunidades da proteína.