The authors declare that they have no competing financial interests.

1. Purification of CFTR
NOTE: Please see Table of Materials and Equipment for a list of equipment and materials used in this protocol. A detailed protocol exists for overexpression of human Wt-CFTR and mutants in the Sf9-baculovirus expression system17,28. Overexpress CFTR and prepare pellets of Sf9 cells according to this protocol.
<ol>
	<li>Crude membrane preparation
	<ol>
		<li>Obtain a fresh or thaw a frozen Sf9 cell pellet from a 500 ml culture of cells over-expressing wildtype-, F508del-, or G551D-CFTR protein with a C-terminal His10-tag, grown by standard cell culture methods, as described previously17,28. Cell pellets will have a volume of approximately 5 ml and a wet weight of approximately 5 g.</li>
		<li>Resuspend Sf9 cells in 20 ml of phosphate buffered saline (PBS) pH 7.4 with protease inhibitor cocktail (1 tablet per 50 ml) at 4 &#176;C, ensuring the sample appears visually homogeneous and no clumps of cells remain.</li>
		<li>Lyse cells using a high pressure cell disruptor (Table of Materials and Equipment), reaching a minimum of 10,000 psi (preferably 15,000-20,000 psi) for 2 min per passage. Keep the sample at 4 &#176;C during lysis.
		<ol>
			<li>Collect the sample in a tube on ice after the lysis period then immediately reload the sample in the cell disruptor. Repeat lysis procedure 3 times. Examine under a microscope to ensure less than 10% remain intact. 
			NOTE: other methods to lyse cells, such as glass beads, etc. have not been rigorously examined for this system, however a user may find such an alternate method suitable.</li>
		</ol>
		</li>
		<li>Centrifuge cell lysis material at 4 &#176;C in a high speed centrifuge at 2,000 x g for 20 min. Collect the supernatant and dispose of the pellet. Divide the supernatant among 20 tubes and centrifuge the samples for 1 hr at 4 &#176;C at 125,000 x g in a table top ultracentrifuge.</li>
		<li>Remove and discard the supernatant, being careful not to disturb the pellet, and store pellets in the same tubes at -80 &#176;C for less than 2 months until use, or retain on ice and continue with the purification immediately.</li>
	</ol>
	</li>
	<li>CFTR solubilization
	<ol>
		<li>Obtain 1-4 fresh or frozen Sf9 crude membrane pellets and resuspend each in 1 ml of Buffer 1 (2% fos-choline; see Table 1 for complete recipe) at 4 &#176;C on ice, using a 25 G needle and 1 ml syringe until the solution is homogeneous by eye without visible cell membrane clumps. Where more than one pellet is being solubilized, continue to treat each sample per the instructions for a single pellet below in parallel, pooling the samples at step 1.3.2.</li>
		<li>Dissolve 1 mini protease inhibitor tablet in 1 ml of Buffer 2 (10 mM imidazole; see Table 1) which lacks fos-choline 14 detergent (protease inhibitors are 10x more concentrated than their recommended dilution at this point) and add 100 &#181;l to the resuspended crude membrane pellet (protease inhibitors are at their recommended dilution at this point). Set on ice for 45-60 min with mixing by inversion 5 times every 5 min.</li>
		<li>Centrifuge resuspended crude membrane pellet in a table top ultracentrifuge for 25 min at 125,000 x g and 4 &#176;C. Retain the supernatant, which contains solubilized CFTR and discard the pellet.</li>
	</ol>
	</li>
	<li>Column binding, phosphorylation and elution
	<ol>
		<li>Wash 400 &#181;l of nickel-NTA (nitrilotriacetic acid) agarose slurry or equivalent resin with 1 ml of Buffer 2 (10 mM imidazole; see Table 1) which lacks detergent, to remove solvent and buffer at 4 &#176;C. Repeat wash twice more.</li>
		<li>Combine solubilized CFTR, remaining protease inhibitor (900 &#181;l) and nickel resin (from 400 &#181;l slurry) to a total of 10 ml with Buffer 2 (10 mM imidazole; see Table 1) which lacks any added detergent. The fos-choline 14 concentration is effectively diluted to 0.2% (w/v) at this step. If there are multiple tubes, pool all samples containing solubilized CFTR, protease inhibitors, nickel-NTA resin and 0.2%(w/v) fos-choline-14 at this point. Incubate for 60 min at 4 &#176;C with gentle shaking.</li>
		<li>Pass CFTR-protease inhibitor-nickel NTA solution down a small screening column (Table 1) or an equivalent empty column.</li>
		<li>Wash the nickel NTA resin, which is retained in the column, with 4 ml per initial pellet of Buffer 3 (10 mM imidazole+DDM; see Table 1) which lacks fos-choline 14 but contains 1 mM DDM (dodecyl maltoside detergent), at 4 &#176;C . Addition of DDM detergent does not significantly alter the pH of this buffer.</li>
		<li>Prepare PKA-MgATP solution by adding 25 &#181;l (5 mM, final concentration) of MgATP, 2,500 U of cAMP-dependent protein kinase A, catalytic subunit (PKA) to 475 &#181;l of Buffer 3 (10 mM imidazole+DDM; see Table 1) to the column. Phosphorylate protein by sealing the bottom end of the column with a cap or Parafilm, and adding 500 &#181;l of PKA-MgATP solution for each initial pellet used.</li>
		<li>Incubate at room temperature (20 &#176;C) for 20 min with gentle mixing and resuspension of the resin using a 1 ml pipettor every 2 min to ensure that PKA has come in contact with the entire surface of the nickel NTA resin.</li>
		<li>Remove the cap and elute the PKA/MgATP solution from the column. Wash the column with 2 ml of Buffer 3 (10 mM imidazole+DDM; see Table 1) at 4 &#176;C.</li>
		<li>Multiply the number of pellets used in the experiment by 4. Wash the column with this number of ml of Buffer 4 (50 mM imidazole+DDM; see Table 1) at 4 &#176;C, by adding 4 ml of buffer to the column, allowing it to flow through and immediately adding the next 4 ml aliquot to the column.</li>
		<li>Elute the protein by passing 1 ml of Buffer 5 (600 mM imidazole+DDM; see Table 1) at 4 &#176;C per initial pellet used through the column, 1 ml at a time without a pause to allow the column to dry, and collect the entire eluate containing purified CFTR in a single tube.</li>
		<li>Concentrate protein sample to remove imidazole and low molecular weight degradation products using a centrifuge filter device (100,000 molecular weight cut-off) such as described in Table of Materials and Equipment or other similar device, in a total of 10 ml Buffer 6 (75 mM KI+DDM; see Table 1) or other buffer of choice containing 1 mM DDM (such as Buffer 7 for non-iodide efflux experiments, 25 mM HEPES+DDM; see Table 1), by centrifuging at 2,000 x g and 4 &#176;C for about 20 min until the sample concentrates to 200 &#181;l. Dilute sample to 10 ml and repeat the concentration step. 
		NOTE: In our experience (unpublished), imidazole significantly inhibits the ATPase activity of CFTR and should be removed at this step by dilution and concentration at least twice if the sample will be used for functional measurements.</li>
		<li>Collect concentrated purified CFTR. Keep at 4 &#176;C in the presence of DDM buffer until use within 1-2 days or continue on immediately to the reconstitution step. Do not freeze the purified protein, as in our experience we observe reduced activity.</li>
	</ol>
	</li>
</ol>
2. Reconstitution of CFTR
<ol>
	<li>Lipid preparation 
	NOTE: CFTR has been successfully reconstituted into Egg PC, POPC, 2:1 (w/w) Egg PC:POPC (1:1 w/w) mixture, or a mixture of PE:PS:PC:ergosterol, 5:2:2:1 (w/w).
	<ol>
		<li>For iodide efflux experiments, dry 5 mg of Egg PC (200 &#181;l of 25 mg/ml stock, see Table of Materials and Equipment) under a stream of argon gas for 20 min at room temperature in a Pyrex or other sturdy (non-borosilicate) glass tube.</li>
		<li>Using absorbance at 280 nm, an ELISA assay or other method, estimate the concentration of the purified CFTR protein sample. For iodide efflux experiments with wildtype CFTR, use a protein:lipid ratio of 1:300-1:3,000 (w/w) to produce a sufficient signal. For G551D- and F508del-CFTR, use a protein:lipid ratio of approximately 1:200-1:1,200 (w/w) in order to detect efflux activity.</li>
		<li>Optimize the protein:lipid ratio for each particular system. Calculate the volume of purified protein required. Calculate the volume of buffer with 1 mM DDM required to make a final volume of 1 ml, i.e., 1 ml &#8211; (volume of protein solution required) = volume of buffer.
		<ol>
			<li>Add the calculated volume of the desired buffer system with 1 mM DDM to the dried lipid (but do not add the CFTR protein at this time) and cover the glass tube with Parafilm. For iodide efflux experiments, resuspend lipids in Buffer 6 (75 mM KI+DDM; see Table 1). For other experiments resuspend in Buffer 8 (25 mM HEPES+DDM, see Table 1) or another desired internal buffer containing 1 mM DDM.</li>
			<li>Vortex for 2 min at room temperature to aid in suspension of the lipid in the DDM buffer. Alternately, heat the sample to 37 &#176;C for 1-2 min before vortexing.</li>
		</ol>
		</li>
		<li>Suspend the lipid repeatedly at room temperature with a 1 ml syringe and 25 G needle until no lipid film is visible on the sides of the tube. Avoid injecting air into the solution which would produce fine bubbles and aid in oxidation of iodide and lipids.</li>
		<li>Sonicate the suspended lipid in the Parafilm covered tube for a 20 sec burst in a bath sonicator containing ice, and then transfer immediately to ice for 1 min. Repeat 3 times. 
		NOTE: Intense sonication turns iodide solutions yellow due to oxidation. Therefore avoid excessive sonication. Monitor the sample for color changes. If a color change is noted, discard the sample and prepare again. The change in color due to oxidation of some of the iodide would result in oxidation of the lipids under the same conditions. Displacing the air from the tube with argon gas before sonicating the solution will help prevent oxidation of lipids and iodide. Intense sonication can break poor quality or scratched glass tubes resulting in loss of the sample.</li>
		<li>Add the volume of purified CFTR calculated in step 2.1.3 to the sonicated lipid sample to a achieve a final volume of 1 ml. Mix the protein with the lipid and detergent solution by resuspension 10 times with a 25 G needle and 1 ml syringe.</li>
		<li>Set lipid and protein sample on ice for 30 min with swirling of tube to mix 5 times every 5 min.</li>
	</ol>
	</li>
	<li>Detergent-binding column preparation
	<ol>
		<li>Obtain a single use commercial detergent-binding spin column (see Table of Materials and Equipment) with a 1 ml volume capacity and break the bottom tab to start the column flowing.</li>
		<li>Centrifuge the column for 2 min at 2,000 x g to remove the storage buffer, and discard this buffer.</li>
		<li>Add 5 ml of Buffer 7 (75 mM KI, see Table 1) to the column and then centrifuge at 200 x g for 4 min to elute the wash buffer. Repeat this step 2 more times for a total of 3 washes to remove all traces of the storage buffer. Discard all flow-through. 
		NOTE: It is critical that the buffer used to wash the column DOES NOT contain DDM or any other detergent. The column has a finite binding capacity for detergent and if a buffer containing detergent is used, the column will be ineffective at removing the detergent from the protein-lipid-detergent sample.</li>
		<li>Carefully aliquot all 1 ml of the lipid-detergent-protein sample onto the top of the column resin and incubate sample at the top of the column for 2 min at room temperature.</li>
		<li>Centrifuge the sample at room temperature for 4 min at 2,000 x g and collect the cloudy proteoliposome sample in a clean 1.5 or 2 ml microfuge tube or a glass tube and place the sample on ice.</li>
		<li>Collect remaining proteoliposomes in the column by adding 200 &#181;l of Buffer 7 (75 mM KI, Table 1) or other buffer (without detergent) to the top of the column and repeat the centrifugation step for 2 min.</li>
		<li>Add the second eluate to the proteoliposome sample if it looks cloudy.</li>
		<li>Store the sample at 4 &#176;C overnight or use immediately for iodide efflux measurements.</li>
	</ol>
	</li>
</ol>
3. Iodide Efflux Measurements for Purified, Reconstituted CFTR
<ol>
	<li>Generation of an iodide gradient across the proteoliposomal membrane
	<ol>
		<li>Pre-swell Sephadex G50 beads in Buffer 9 (75 mM K-Glu; potassium glutamate, see Table 1), (approximately 5 g dry beads in 50 ml buffer) or desired external buffer at room temperature for at least 2 hr or at 4 &#176;C overnight.</li>
		<li>Prepare 4 Sephadex G50 columns saturated in Buffer 9 (75 mM K-Glu, see Table 1) or other buffer in small screening columns by loading resin to at least &#190; full in the column.</li>
		<li>Centrifuge for 4 min at 2,000 x g to remove excess buffer from the resin. There should be approximately 2.25 ml of packed hydrated resin in the column at the end of the centrifugation step. 
		NOTE: Resin should be lightly hydrated but not immersed in liquid and a subsequent brief spin should not elute significant volumes of buffer. It is critical to the successful elution of the proteoliposome sample that the column resin is neither too wet nor too dry and the user may need to experiment with the columns in order to become familiar with the most successful buffer exchange conditions.</li>
		<li>Carefully add 125-150 &#181;l of proteoliposome sample to the top of each column and centrifuge for 4 min at 2,000 x g.</li>
		<li>Pool proteoliposome eluates together for immediate use in iodide efflux or other experiments. 
		NOTE: The eluted volume from each column should be roughly 150 &#181;l and the sample should be somewhat cloudy, but less cloudy than the original sample. Larger volumes or clear eluates suggest that the columns haven&#8217;t been dried sufficiently, or have been dried too much, respectively, and will not result in a successful iodide efflux experiment.</li>
	</ol>
	</li>
	<li>Iodide efflux assay
	<ol>
		<li>&#160;Wash an iodide selective micro electrode (Table of Materials and Equipment) extensively with de-ionized water, and then incubate for 20 min before the experiment in Buffer 10 (15 &#181;M KI, see Table 1). Wash the electrode again extensively with de-ionized water.</li>
		<li>Add 150 &#181;l of drug (20 &#181;M typical concentration to produce a final concentration of 10 &#181;M in the assay) in Buffer 9 (75 mM K-Glu, see Table 1) or vehicle in Buffer 9 to one well of a 96-well plate with a small flea stir bar.
		<ol>
			<li>Ensure that there are no bubbles present. Use a pipette tip to remove stray bubbles. If a drug solution is being used, ensure that the final DMSO concentration in the well is less than 1% (v/v) (typically 0.1-0.2% (v/v)).</li>
		</ol>
		</li>
		<li>Add 150 &#181;l of reconstituted CFTR protein that was produced in section 3.1 in Buffer 9 (75 mM K-Glu, see Table 1) to the well with drug or buffer, to produce a total volume of 300 &#181;l in the well of the plate.</li>
		<li>Place the 96-well plate on a stir plate and stir at 130 rpm (or at a moderate speed of approximately &#188; of the maximum speed).</li>
		<li>Insert the tip of the iodide selective electrode carefully into the well with the sample such that the tip is not touching the bottom of the well or being hit by the stir bar. Ensure that the reference electrode, if separate, is also in contact with the solution in the well.</li>
		<li>Record tracings using a home-made or commercial computer program that interfaces with the electrode (see Table of Materials and Equipment for details). Use a sampling rate of 2 Hz, with filtering of the signal through a low pass filter.</li>
		<li>Allow the sample to equilibrate for 5 min to produce a stable flat, or near flat baseline while recording.</li>
		<li>Add 3 &#181;l MgATP (100 mM stock prepared in dH2O and adjusted to pH 7 with KOH; 1 mM final concentration) to the sample to activate the protein. Allow ~5 min to reach a new stable baseline. Always adjust the pH of the 100 mM MgATP stock to neutral before use to avoid changes in the pH of the external buffer.</li>
		<li>Add 3 &#181;l of valinomycin stock (2 &#181;M; 20 nM final concentration) to the sample to shunt changes in potential difference generated by iodide-selective conductance through CFTR. Record the decreasing slope (decrease in mV) due to CFTR-mediated iodide efflux activity for at least 5 min.</li>
		<li>To ensure that the trapping of iodide in the proteoliposome lumen was sufficient, add 3 &#181;l of 10% (v/v) Triton X-100 to the sample. Significant and immediate iodide release indicates that sufficient iodide has been trapped in the vesicles such that trapping was not the limiting factor for the changes in slope determined in 3.2.9 but rather the CFTR-mediated activity.</li>
		<li>Wash the electrode and stir bar thoroughly with de-ionized water after treatment of samples with drug or with Triton X-100 to ensure all traces of these reagents have been removed to avoid contamination of the next sample.</li>
		<li>Prepare a series of dilute KI concentrations in the micromolar to nanomolar range in Buffer 9 (K-Glu buffer, see Table 1). Record the mV response of the electrode to each concentration from 0 to the highest concentration prepared. Construct a standard curve where the probe response (mV) is plotted vs. log of the molar iodide concentration. Use the standard curve to convert the mV response of the probe during the efflux experiment to concentration of iodide released. 
		NOTE: Prepare a calibration curve on a weekly basis until a user is sure of how quickly the response of the probe changes. There will be a drift in the response and sensitivity of the probe over time. As the probe ages, the response will degrade. This may be partially abrogated by changing the internal solutions periodically and extensive washing of the probe tip in water after use, which likely limits adherence of molecules to the probe surface.</li>
		<li>Determine the average slope of the line of the concentration vs. time plot for each proteoliposome sample for the last 30 sec before addition of MgATP, and after addition of valinomycin but before addition of Triton X-100. Subtract the baseline slope from the valinomycin to determine the CFTR-mediated iodide efflux activity for that sample.</li>
	</ol>
	</li>
</ol>

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D., 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=VX-809+and+Related+Corrector+Compounds+Exhibit+Secondary+Activity+Stabilizing+Active+F508del-CFTR+after+Its+Partial+Rescue+to+the+Cell+Surface.">VX-809 and Related Corrector Compounds Exhibit Secondary Activity Stabilizing Active F508del-CFTR after Its Partial Rescue to the Cell Surface.</a> Chem Biol. 21 (5), 666-678 (2014).</li><li>Kim Chiaw, P., Wellhauser, L., Huan, L. J., Ramjeesingh, M., Bear, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+corrector+modifies+the+channel+function+of+F508del-CFTR.">A chemical corrector modifies the channel function of F508del-CFTR.</a> Mol.Pharmacol. 78 (3), 411-418 (2010).</li><li>Verkman, A. S., Galietta, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+channels+as+drug+targets.">Chloride channels as drug targets.</a> Nat Rev Drug Discov. 8 (2), 153-171 (2009).</li><li>Pasyk, S., Li, C., Ramjeesingh, M., Bear, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+interaction+of+a+small-molecule+modulator+with+G551D-CFTR,+a+cystic+fibrosis-causing+mutation+associated+with+severe+disease.">Direct interaction of a small-molecule modulator with G551D-CFTR, a cystic fibrosis-causing mutation associated with severe disease.</a> Biochem J. 418 (1), 185-190 (2009).</li><li>Norez, C., 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=Determination+of+CFTR+chloride+channel+activity+and+pharmacology+using+radiotracer+flux+methods.">Determination of CFTR chloride channel activity and pharmacology using radiotracer flux methods.</a> J Cyst Fibros. 3 (2), 119-121 (2004).</li><li>Whitney, J. C., 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=Structural+basis+for+alginate+secretion+across+the+bacterial+outer+membrane.">Structural basis for alginate secretion across the bacterial outer membrane.</a> Proc Natl Acad Sci U S A. 108 (32), 13083-13088 (2011).</li><li>Islam, S. T., 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=Proton-dependent+gating+and+proton+uptake+by+Wzx+support+O-antigen-subunit+antiport+across+the+bacterial+inner+membrane.">Proton-dependent gating and proton uptake by Wzx support O-antigen-subunit antiport across the bacterial inner membrane.</a> mBio. 4 (5), e00678-e00613 (2013).</li><li>Ramjeesingh, M., Conn, P. 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=Ch.+16.">Ch. 16.</a> Reliable Lab Solutions: Essential Ion Channel MethodsReliable Lab Solutions. , 337-357 (2010).</li><li>Wellhauser, L., 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+small-molecule+modulator+interacts+directly+with+deltaPhe508-CFTR+to+modify+its+ATPase+activity+and+conformational+stability.">A small-molecule modulator interacts directly with deltaPhe508-CFTR to modify its ATPase activity and conformational stability.</a> Mol Pharmacol. 75 (6), 1430-1438 (2009).</li><li>Lee, S. Y., Letts, J. A., 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+reconstitution+of+purified+human+Hv1+H++channels.">Functional reconstitution of purified human Hv1 H+ channels.</a> J Mol Biol. 387 (5), 1055-1060 (2009).</li><li>Chang, X. 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=Protein+kinase+A+(PKA)+still+activates+CFTR+chloride+channel+after+mutagenesis+of+all+10+PKA+consensus+phosphorylation+sites.">Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites.</a> J. Biol. Chem. 268 (15), 11304-11311 (1993).</li><li>Gadsby, D. C., Nairn, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+CFTR+Cl-+ion+channels+by+phosphorylation+and+dephosphorylation.">Regulation of CFTR Cl- ion channels by phosphorylation and dephosphorylation.</a> Adv.Second Messenger Phosphoprotein Res. 33, 79-106 (1999).</li><li>Li, C., 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=ATPase+activity+of+the+cystic+fibrosis+transmembrane+conductance+regulator.">ATPase activity of the cystic fibrosis transmembrane conductance regulator.</a> J Biol Chem. 271 (45), 28463-28468 (1996).</li><li>Ma, T., 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=Thiazolidinone+CFTR+inhibitor+identified+by+high-throughput+screening+blocks+cholera+toxin-induced+intestinal+fluid+secretion.">Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion.</a> J Clin Invest. 110 (11), 1651-1658 (2002).</li><li>Kopeikin, Z., Sohma, Y., Li, M., Hwang, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanism+of+CFTR+inhibition+by+a+thiazolidinone+derivative.">On the mechanism of CFTR inhibition by a thiazolidinone derivative.</a> J Gen Physiol. 136 (6), 659-671 (2010).</li><li>Wang, X. F., Reddy, M. M., Quinton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+new+cystic+fibrosis+transmembrane+conductance+regulator+inhibitor+on+Cl-+conductance+in+human+sweat+ducts.">Effects of a new cystic fibrosis transmembrane conductance regulator inhibitor on Cl- conductance in human sweat ducts.</a> Exp Physiol. 89 (4), 417-425 (2004).</li></ol>

The authors acknowledge the advice of Dr. Mohabir Ramjeesingh during development of the purification methods described here. These studies were supported by Operating Grants to C.E.B. from the Canadian Institutes of Health Research (CIHR) and Cystic Fibrosis Canada (CFC). P.D.W.E. was supported in part by Fellowship awards through the CIHR and CFC. The authors acknowledge the provision of corrector, potentiator and inhibitor compounds from Dr. R. Bridges (Rosalind University of Medicine and Science) and distribution by the Cystic Fibrosis Foundation Therapeutics (CFFT). The authors also acknowledge outstanding service by the Baylor Cell Culture Facility (NIH grant P30 CA125123) in expressing Wt and mutant CFTR protein using the baculovirus system. The inactive VX-770 analog, V-09-1188, was a gift of Vertex Pharmaceuticals.

There have been a limited number of purification protocols for full-length, functional CFTR isolation, from a variety of cellular overexpression systems. The method described here is advantageous as it allows rapid purification of Wt-CFTR or high enrichment of F508del- and G551D-CFTR in moderate quantities that is highly functional in assays including ATPase and direct measurements of channel function, including single channel measurements in planar bilayer systems and demonstrated measures of CFTR population channel fun...

Chloride transport across the apical membranes of epithelial cells in such tissues as the lung, intestine, pancreas and sweat glands is primarily mediated by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), an ATP- and phosphorylation-regulated member of the ABC (ATP-Binding Cassette) C subfamily of membrane proteins (reviewed in1). Like other members of the ABCC subfamily, CFTR is a large, multi-spanning integral membrane protein that binds ATP at two nucleotide binding sites formed at the interface of its nucleotide-binding domains (NBDs), where it possesses modest ATPase activity at a single site. However, unlike other ABCC subfamily members, CFTR has evolved as a unique regulated Cl channel rather than as an active solute transporter.
Mutations in CFTR cause Cystic Fibrosis, a disease affecting multiple organs including the lungs, gastrointestinal tract, pancreatic and reproductive tract, leading to morbidity and mortality in young adults. Lung disease typically accounts for early mortality in Cystic Fibrosis and in most cases is caused by the loss of CFTR function on the surface epithelium of the conducting airways. The lack of CFTR chloride channel activity causes a reduction in both Cl- and water movement across the surface epithelium to modify the fluid layer on the apical surface of the ciliated respiratory epithelium. This results in a viscous airway surface liquid that impairs the ability of ciliated respiratory epithelial cells to effectively clear pathogens out of the airways. As a consequence, most CF patients suffer from recurrent bouts of lung infection and lung damage due to inflammation.
As expected, studies of the mechanism of action of the normal CFTR protein focused primarily on detailed electrophysiological studies of its channel gating activity. Single channel studies have shown directly that CFTR functions as a PKA-dependent Cl- channel which possesses an ATP regulated gate2. Detailed electrophysiological studies provide a great deal of information on single CFTR channels1,3, however there may be concern as to whether the characteristics of any particular single channel that has been studied is reflective of the entire population of CFTR channels and therefore the single channel results should always be considered along with methods to study the macroscopic population. Direct assay of the population channel activity of purified CFTR has the potential to provide insight into the molecular defect associated with disease-causing mutations and to drive discovery of chemical modulators which repair mutant CFTR proteins. To date, there are in excess of 1,900 different mutations in CFTR thought to cause Cystic Fibrosis4. The major mutation, F508del-CFTR, found on at least one allele in approximately 90% of patients in North America and Europe leads to protein misfolding and retention in the endoplasmic reticulum5. F508del-CFTR also has other consequences, including defective channel activity6-9. The resultant absence of CFTR from the cell surface is associated with severe disease. G551D-CFTR, a less common mutation, is thought to be properly folded yet is dysfunctional as a chloride channel at the cell surface6. The development of small molecule correctors and potentiators has the goal of correcting folding and/or trafficking of mutants such as F508del-CFTR to the cell surface, and potentiating or increasing the channel activity of mutations such as G551D when present on the cell surface, respectively. While the correctors VX-809 and VX-661 (are not yet approved for use in patients, the potentiator Kalydeco (ivacaftor; VX-770) is being used at 150 mg every 12 hr in CF patients &#62;6 years with at least one G551D-CFTR mutation, and more recently for patients with one of G178R, S549N, S549R, G551S, G1244E, S1251N, S1255P and G1349D. Kalydeco is both safe and results in improvement of clinical measures of CF disease10, however the mechanism of action of this small molecule was poorly understood at the time of FDA approval for use in patients.
A handful of CFTR purification methods have been described previously2,11-18, many of which require a considerable length of time to complete. In a recent publication19, a unique rapid purification and reconstitution method was described for CFTR overexpressed in the Sf9 cell expression system, and this purified protein in defined lipid systems was used to develop a CFTR halide channel activity assay for a population of CFTR molecules. The assay recapitulates the known effects of phosphorylation, nucleotides and inhibitors on CFTR function. The system was used to interrogate the effects of the potentiator VX-770/Kalydeco on Wt- (wild-type), F508del- and G551D-CFTR and it was shown for the first time that the drug interacts directly with the CFTR protein to potentiate its channel activity in an ATP-independent manner, demonstrating the utility and applicability of these methods to the study of the interaction of CFTR and mutants with nucleotides and small molecules from a population perspective to answer clinically relevant questions about the protein. The methods have also been used to study other potentiator molecules and their derivatives20, as well as the effects of a small molecule corrector on the activity of the protein21.
Efflux assays have been used in many studies previously to investigate the activity of CFTR mutants and the effects of CFTR-modulatory compounds on its activity, including whole cell assays using electrodes, radioactive tracers and fluorophores22,23, membrane vesicles with ion selective electrodes24, and purified reconstituted CFTR with radioactive tracers25. However the use of ion selective electrodes to study purified reconstituted CFTR was first reported recently19. An adaptation of the current method has been used for reconstitution and functional characterization of two membrane proteins in Pseudomonas aeruginosa, a common CF pathogen. Reconstitution of purified AlgE outer membrane protein coupled with iodide efflux measurements were used to support a model for anionic alginate secretion through this transporter26. Reconstitution and iodide efflux measurements were applied to the purified Wzx protein, which allowed a model to be proposed that suggests an H+-dependent antiport mechanism for lipid-linked oligosaccharide translocation across the bacterial inner membrane by this protein27. In both cases iodide was used as a surrogate for the anionic substrate, albeit at lower throughput than one might expect for a native substrate. The method may be suitable for adaptation to other proteins with cationic transport or conduction pathways for anionic substrates.
Here a rapid purification procedure is described for the CFTR protein and its reconstitution into proteoliposomes of defined lipid. The rapid reconstitution procedure can easily be tailored for use with CFTR purified by other methods, provided that the type of detergent used in the purification is amenable to removal by the methods used here or can be exchanged for a suitable detergent before the reconstitution procedure. The iodide efflux method for measurement of channel activity of purified and reconstituted CFTR protein is described in detail and some typical results that can be obtained from this method are presented.

<table border="0" cellpadding="0" cellspacing="0" width="1269">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:403px;">fos-choline 14 detergent</td>
			<td style="width:515px;">Anatrace Affymetrix (www.anatrace.affymetrix.com)</td>
			<td style="width:145px;">F312</td>
			<td style="width:207px;">Affymetrix: Anatrace Products; CAS#&#160;:77733-28-9</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cOmplete EDTA-free protease inhibitor cocktail tablets</td>
			<td>Roche (www.roche-applied-science.com)</td>
			<td>04 693 132 001</td>
			<td>EDTA-free Protease inhibitor cocktail tablets (1 in 50 ml or mini:1 in 10 mL) (Roche Diagnostics GmbH; Ref: 11873 580 001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack&#160;</td>
			<td>Roche (www.roche-applied-science.com)</td>
			<td>05 892 791 001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ni-NTA Agarose</td>
			<td>Qaigen GmbH</td>
			<td align="right">1018240</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fisherbrand Screening columns</td>
			<td>Fisher Healthcare</td>
			<td>11-387-50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amicon Ultra Centrifugal filters, Ultracel-100K</td>
			<td>Millipore (www.millipore.com)</td>
			<td>UFC910008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cAMP-Dependent Protein Kinase A (PKA), Catalytic Subunit</td>
			<td colspan="2">New England Biolabs (www.neb.com/products/p6000-camp-dependent-protein-kinase-pka-catalytic-subunit)</td>
			<td>Peirce PKA is also suitable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PC, Chicken Egg</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>840051C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">POPC</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PS, Porcine Brain</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>840032C</td>
			<td>(CFTR has been successfully reconstituted into Egg PC, POPC, 2:1 (w/w) Egg PC:POPC, or a mixture of PE:PS:PC:ergosterol, 5:2:2:1 (w/w))</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PE, Chicken Egg</td>
			<td>Avanti Polar Lipids (www.avantilipids.com)</td>
			<td>841118C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ergosterol</td>
			<td>Sigma (www.sigmaaldrich.com)</td>
			<td align="right">45480</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce Detergent removal spin column</td>
			<td>Thermo Scientific</td>
			<td align="right">87779</td>
			<td>1 ml capacity columns</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">valinomycin</td>
			<td>Sigma (www.sigmaaldrich.com)</td>
			<td>V-0627</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VX-770 (ivacaftor)</td>
			<td>Selleck Chemicals</td>
			<td>S1144</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iodide selective microelectrode</td>
			<td>Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/microionn.tam)</td>
			<td>LIS-146ICM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clampex 8.1 software</td>
			<td>Axon Instruments (www.axon.com)</td>
			<td></td>
			<td>we use components of the ClampX system with a home made filter to monitor and record the response to our electrode</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alternate Software: ArrowLabb System&#160;</td>
			<td>Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/ionsystems.tam)</td>
			<td>LIS-146LICM-XS</td>
			<td>Lazar Research sells a meter that can interface with a computer and software to record the probe response.&#160; This software should serve a similar function to our setup</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">small stir bars</td>
			<td>Big Science Inc (www.stirbars.com)</td>
			<td>SBM-0502-CMB</td>
			<td>choose a stir bar small enough to easily fit into a well of a 96-well plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sephadex G50, fine</td>
			<td>GE Health Care (www.gelifesciences.com)</td>
			<td>17-0042-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sonicator</td>
			<td>Laboratory Supplies Co, Inc.</td>
			<td>G112SP1G</td>
			<td>Bath sonicators from other manufacturers should also be suitable</td>
		</tr>
	</tbody>
</table>

functional reconstitution and channel activity measurements of purified wildtype and mutant cftr protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described in this written publication are methods to purify, reconstitute and measure regulated channel activity of the CFTR protein. Figure 1a shows the workflow for the purification, reconstitution and iodide efflux procedures. Methods for reconstitution and channel activity measurements by iodide flux are also shown in further detail in the associated video.
Purification and reconstitution of CFTR into proteoliposomes
CFTR can be functionally exp...

Watch this Scientific Journal Video about Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein at JoVE.com

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

functional-reconstitution-channel-activity-measurements-purified

Research

JoVE Journal

Biology

12.1K vues.  Hospital for Sick Children.  Décrit ici est un procédé rapide et efficace pour la reconstitution fonctionnelle de type sauvage purifiée et une protéine de CFTR mutante qui conserve l'activité de ce canal de chlorure, qui est défectueux dans la fibrose kystique. Efflux d'iodure de protéoliposomes reconstituées médiées par CFTR permet des études de l'activité du canal et les effets de petites molécules. 

Vidéo: Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

Biochemical Reconstitution of Steroid Receptor&amp;#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

The method presented here describes an assay to follow activation of RhoA specific GDP/GTP Exchange Factors (GEFs) in cultured cells by making use of a mutant RhoA GST fusion protein that has high affinity for activated GEFs. GEFs are precipitated from cell lysates, detected by Western blotting and quantified by densitometry.

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

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 ...

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel&#8217;s &#947;-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (&#916;Iami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of &#947;ENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a &#947;ENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in &#947;ENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel ENaC by Combining Current Measurements with Detection of Cleavage Fragments

Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis.

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously ...

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...