We thank Michael J. Bruno, Radda Rusinova and Jon T. Sack for many stimulating discussions. Financial support from NIH, R01GM021342 and ARRA supplement R01GM021342-35S1, and the Josiah Macy, Jr. Foundation to OSA; the Tri-I CMB program for HII; and The Iris L. and Leverett S. Woodworth Medical Scientist Fellowship and NIH MSTP grant GM07739 for RK.

1. Generate ANTS-filled Liposomes
<ol>
 <li>On day 1, remove organic solvent from lipids.</li> 
	<li>Remove lipid from freezer and let it equilibrate to room temperature.</li> 
	<li>Add 0.6 mL of 25 mg/mL (1,2-dierucoyl-sn-glycero-3-phosphocholine) lipid in chloroform solution to a 25 mL round bottom flask.</li> 
	<li>Continually rotate the flask while drying under nitrogen, until all the chloroform has evaporated and a thin white film of lipid coats the entire lower half of the flask.</li> 
	<li>Dry in a des...

<ol>
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We have demonstrated a fast fluorescence-based assay for determining the bilayer modifying potential of drugs and other small amphiphiles. Compounds that modify bilayer properties are likely to alter membrane protein function in an indirect, nonspecific manner, possibly contributing to "off-target" drug effects. The assay exploits the power of gramicidin channels as probes for changes in bilayer properties 12 that are sensed by bilayer-spanning proteins. The results obtained using the fluorescence-based ass...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>ANTS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-350</td>
<td>&nbsp;</td>
<tr>
<td>gramicidin</td>
<td>&nbsp;</td>
<td>Sigma Chemical Co</td>
<td>G-5002</td>
<td>&nbsp;</td>
<tr>
<td>1,2-dierucoyl-sn-glycero-3-phosphocholine</td>
<td>&nbsp;</td>
<td>Avanti Polar Lipids</td>
<td>850398C</td>
<td>&nbsp;</td>
<tr>
<td>Mini-Extruder kit</td>
<td>&nbsp;</td>
<td>Avanti Polar Lipids</td>
<td>610000</td>
<td>&nbsp;</td>
<tr>
<td>PD-10 Desalting column</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich Made by GE Healthcare</td>
<td>54805</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

gramicidin-based fluorescence assay; for determining small molecules potential for modifying lipid bilayer properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

Watch this Scientific Journal Video about Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties at JoVE.com

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

gramicidin-based-fluorescence-assay-for-determining-small-molecules

Research

JoVE Journal

Biology

12.7K visualizações.  Weill Cornell Medical College. Nós introduzimos um ensaio de fluorescência baseada rápido que monitora a taxa de extinção de fluorescência como uma medida da atividade do canal gramicidin. Os canais gramicidin são usados ​​como transdutores de força molecular para monitorar as mudanças nas propriedades bicamada lipídica como sentiu por proteínas bicamada de expansão.