pAnap was a kind gift from Dr. Peter Schultz (Scripps Research Institute). This work was funded by the Canadian Institutes for Health Research Grants MOP-102689 and MOP-136894 (to R.B.) and Canadian Foundation for Innovation Grant 950-225005.

Frog manipulations were performed in accordance with the Canadian guidelines and have been approved by the ethics committee (CDEA, protocol #15-042) of University of Montr&#233;al.
1. mRNA Preparation for fUAA Incorporation
<ol>
	<li>Choose a site of interest in the protein where conformational changes are expected to occur. Select an amino acid in this region to be substituted for the fUAA. 
	NOTE: The choice of position is based on the structural rearrangements that are expected. If a...

<ol>
	<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 (5246), 213-216 (1996).</li><li>Kalstrup, T., Blunck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+internal+pore+opening+in+K(V)+channels+probed+by+a+fluorescent+unnatural+amino+acid.">Dynamics of internal pore opening in K(V) channels probed by a fluorescent unnatural amino acid.</a> Proc Natl Acad Sci U S A. 110 (20), 8272-8277 (2013).</li><li>Xiao, H., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=At+the+Interface+of+Chemical+and+Biological+Synthesis:+An+Expanded+Genetic+Code.">At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code.</a> Cold Spring Harb Perspect Biol. 8 (9), (2016).</li><li>Blunck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+9.">Chapter 9.</a> Handbook of Ion Channels. , 113-133 (2015).</li><li>Chatterjee, A., Guo, J., Lee, H. S., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetically+encoded+fluorescent+probe+in+mammalian+cells.">A genetically encoded fluorescent probe in mammalian cells.</a> J Am Chem Soc. 135 (34), 12540-12543 (2013).</li><li>DeBerg, H. A., Brzovic, P. S., Flynn, G. E., Zagotta, W. N., Stoll, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Energetics+of+Allosteric+Regulation+of+HCN2+Ion+Channels+by+Cyclic+Nucleotides.">Structure and Energetics of Allosteric Regulation of HCN2 Ion Channels by Cyclic Nucleotides.</a> J Biol Chem. 291 (1), 371-381 (2016).</li><li>Shen, 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=Genetically+encoding+unnatural+amino+acids+in+neural+stem+cells+and+optically+reporting+voltage-sensitive+domain+changes+in+differentiated+neurons.">Genetically encoding unnatural amino acids in neural stem cells and optically reporting voltage-sensitive domain changes in differentiated neurons.</a> Stem Cells. 29 (8), 1231-1240 (2011).</li><li>Aman, T. K., Gordon, S. E., Zagotta, W. 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A., Blunck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intersubunit+interaction+between+S4-S5+linker+and+S6+is+responsible+for+the+slow+off-gating+component+in+Shaker+K++channels.">An intersubunit interaction between S4-S5 linker and S6 is responsible for the slow off-gating component in Shaker K+ channels.</a> J Biol Chem. 285 (18), 14005-14019 (2010).</li><li>Kusch, J., 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=How+subunits+cooperate+in+cAMP-induced+activation+of+homotetrameric+HCN2+channels.">How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels.</a> Nat Chem Biol. 8 (2), 162-169 (2012).</li><li>Lee, H. S., Guo, J., Lemke, E. A., Dimla, R. D., Schultz, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+incorporation+of+a+small,+environmentally+sensitive,+fluorescent+probe+into+proteins+in+Saccharomyces+cerevisiae.">Genetic incorporation of a small, environmentally sensitive, fluorescent probe into proteins in Saccharomyces cerevisiae.</a> J Am Chem Soc. 131 (36), 12921-12923 (2009).</li><li>Summerer, 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=A+genetically+encoded+fluorescent+amino+acid.">A genetically encoded fluorescent amino acid.</a> Proc Natl Acad Sci U S A. 103 (26), 9785-9789 (2006).</li><li>Kalstrup, T., Blunck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reinitiation+at+non-canonical+start+codons+leads+to+leak+expression+when+incorporating+unnatural+amino+acids.">Reinitiation at non-canonical start codons leads to leak expression when incorporating unnatural amino acids.</a> Sci Rep. 5, 11866 (2015).</li><li>Liu, H., Naismith, J. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+Xenopus+laevis+and+oocyte+injection.">Maintenance of Xenopus laevis and oocyte injection.</a> Methods Enzymol. 207, 266-279 (1992).</li><li>Rudokas, M. W., Varga, Z., Schubert, A. R., Asaro, A. B., Silva, J. 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+Xenopus+oocyte+cut-open+vaseline+gap+voltage-clamp+technique+with+fluorometry.">The Xenopus oocyte cut-open vaseline gap voltage-clamp technique with fluorometry.</a> J Vis Exp. (85), (2014).</li><li>Zhao, J., Blunck, 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+isolated+voltage+sensing+domain+of+the+Shaker+potassium+channel+forms+a+voltage-gated+cation+channel.">The isolated voltage sensing domain of the Shaker potassium channel forms a voltage-gated cation channel.</a> Elife. 5, (2016).</li><li>Posson, D. J., Ge, P., Miller, C., Bezanilla, F., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+vertical+movement+of+a+K++channel+voltage+sensor+measured+with+luminescence+energy+transfer.">Small vertical movement of a K+ channel voltage sensor measured with luminescence energy transfer.</a> Nature. 436 (7052), 848-851 (2005).</li><li>Chanda, B., Asamoah, O. K., Blunck, R., Roux, B., 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=Gating+charge+displacement+in+voltage-gated+ion+channels+involves+limited+transmembrane+movement.">Gating charge displacement in voltage-gated ion channels involves limited transmembrane movement.</a> Nature. 436 (7052), 852-856 (2005).</li><li>Taraska, J. W., Puljung, M. C., Zagotta, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-distance+probes+for+protein+backbone+structure+based+on+energy+transfer+between+bimane+and+transition+metal+ions.">Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions.</a> Proc Natl Acad Sci U S A. 106 (38), 16227-16232 (2009).</li><li>Baker, B. J., 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=Genetically+encoded+fluorescent+sensors+of+membrane+potential.">Genetically encoded fluorescent sensors of membrane potential.</a> Brain Cell Biol. 36 (1-4), 53-67 (2008).</li><li>Miranda, 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=State-dependent+FRET+reports+calcium-+and+voltage-dependent+gating-ring+motions+in+BK+channels.">State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels.</a> Proc Natl Acad Sci U S A. , (2013).</li><li>Sisido, M., Ninomiya, K., Ohtsuki, T., Hohsaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-base+codon/anticodon+strategy+and+non-enzymatic+aminoacylation+for+protein+engineering+with+non-natural+amino+acids.">Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids.</a> Methods. 36 (3), 270-278 (2005).</li><li>Hohsaka, T., Ashizuka, Y., Murakami, H., Sisido, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five-base+codons+for+incorporation+of+nonnatural+amino+acids+into+proteins.">Five-base codons for incorporation of nonnatural amino acids into proteins.</a> Nucleic Acids Res. 29 (17), 3646-3651 (2001).</li></ol>

The in vivo aminoacylation of tRNAs which are continuously being transcribed together with the tRNA-synthetase, makes it possible to obtain high expression levels for fluorescence measurements. For efficient fUAA incorporation, it is critical that pAnap is correctly injected into the nucleus. Due to the uncertainty of the exact location of the nucleus, 10-40% of the DNA injections are expected to fail, resulting in non-expressing (or leak-expressing) oocytes. Therefore, it is important to check expression in abs...

Over 200 unnatural amino acids of various chemical and physical properties have been genetically incorporated into proteins in E. coli, yeast and mammalian cells3. The unnatural amino acid is incorporated in response to a specific stop codon via an orthogonal engineered tRNA/synthetase pair. The genetical approach to modify proteins has provided valuable insights into protein structure and function. Here, we present a protocol for using Voltage-Clamp Fluorometry (VCF) in combination with a fluorescent UAA.
In VCF, the simultaneous observation of functional data and structural rearrangements localized around the fluorescent probe (~5 &#197;) allows us to obtain dynamic information with millisecond resolution1. The fluorescent probes alter their quenching state upon localized movement of the protein. A movement of only 1-2 &#197; is sufficient to lead to significant changes in the fluorescence intensity4. After identification of the site of interest in the target protein, the site is mutated by point mutation. Classically, the residue had been mutated to a cysteine whereas now, an amber stop codon (TAG) is introduced for genetic fUAA incorporation. The protein is then in vitro transcribed.
While other expression systems (e.g.,&#160;mammalian cells) can be used5,6,7, Xenopus oocytes are preferable for structure-function studies because of their larger size, leading to easier manipulation and higher fluorescence intensity (more fluorophores) and, therefore, to-noise ratio. Furthermore, Xenopus oocytes have low background from endogenous proteins2,8, and the dark pigmentation on the animal pole shields against background fluorescence from the cytosol. The Xenopus oocytes are surgically removed and DNA encoding the orthogonal tRNA/tRNA-synthetase pair specific for the fUAA is injected into the nucleus of the oocytes. After a 6-24 h incubation time, the protein RNA is co-injected with the fUAA into the cytosol of the oocytes, followed by a 2-3 days incubation period. In order to prevent any damage to the fUAA (photobleaching), the procedures including Anap have to be carried out under red light to avoid fluorophore excitation.
Oocytes are studied on a cut-open oocyte voltage-clamp setup, which is mounted on an upright fluorescence microscope, and electrical current and fluorescence changes are simultaneously recorded9,10. Alternatively, two-electrode voltage clamp1 or patch-clamp configurations11can be used. Fluorescence is excited by appropriate wavelengths with low RMS noise and emission recorded using a photodiode linked to an amplifier with high amplification.
There are several advantages of using fluorescent unnatural amino acids (fUAAs) in voltage-clamp fluorometry. One is access to the cytosolic side of the membrane proteins; many regulatory processes are located here (e.g., Ca2+- or nucleotide binding sites, fast and closed-state inactivation of voltage-gated ion channels, pore opening, module coupling). All these processes are now accessible for fluorescent labeling.
Another advantage is the small size of the probe leading to less disturbance of the protein. So far, two orthogonal tRNA/tRNA synthetase pairs for fUAAs have been engineered12,13, where 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap) is the only fUAA which has been used in Xenopus oocytes2,8. Anap is an environmentally sensitive fluorophore with a molecular weight of 272.3 g/mol and is only slightly bigger than tryptophan12 (Figures 1A, 1B). Due to its small size, fewer steric effects are likely to be introduced by the fluorophore compared to conventional fluorophores attached via a linker (typically more than 500 g/mol). Moreover, in the case of Anap, the fluorophore is located closer to the protein backbone than those linked to cysteines, and consequently, Anap is probing more localized rearrangements. Finally, removal of endogenous cysteines in conventional VCF in order to ensure site-specific labeling is no longer a requirement in UAA-VCF and therefore (i) leaves the proteins in (almost) their native state and (ii) allows VCF to be applied to study a wider range of proteins in which function may be altered by cysteine substitution.
<img alt="Figure 1" src="/files/ftp_upload/55598/55598fig1.jpg" /> 
Figure 1: Anap and Fluorescence Spectra. (A) Chemical structure of Anap. (B) Normalized absorption spectrum and emission spectra for 1 nM Anap, demonstrating the sensitivity of Anap fluorescence to the solvent hydrophobicity. Emission spectra were obtained by exciting at 350 nm. <a href="https://www.jove.com/files/ftp_upload/55598/55598fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A disadvantage of using fluorescent UAAs is that a heterogeneous population of proteins may result from stop codon readthrough, translational reinitiation, C-terminal truncated proteins or crosstalk with endogenous aminoacylation if the amount of aminoacylated tRNAs is scarce. Such leak expression should always be checked for in the absence of the fUAA and the tRNA/tRNA synthetase pair. We addressed the issue of translational reinitiation and how to circumvent it for N-terminal insertion sites previously14. However, when the fUAA, tRNA and tRNA synthetase are present in saturated amounts, there only remains a low probability of leak expression.
The key procedural difference between fUAA-VCF and conventional VCF is the injection and handling of the oocytes; the injection of DNA encoding the tRNA and tRNA synthetase (pAnap) is followed by the introduction of Anap, which is either co-injected with the protein mRNA or alternatively added to the incubation solution as an acetoxymethyl (AM) ester.

<table border="0" cellpadding="0" cellspacing="0" width="567">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Solutions</td>
			<td style="width:111px;"></td>
			<td style="width:161px;"></td>
			<td style="width:96px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Barth&#39;s solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>90mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td>3mM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M-9397</td>
			<td>0.82mM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C-7902</td>
			<td>0.41mM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ca(NO3)2</td>
			<td>Sigma-Aldrich</td>
			<td>C-1396</td>
			<td>0.33mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>5mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaOH hydrate</td>
			<td>BDH</td>
			<td>BDH7225-4</td>
			<td>pH 7.6</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicilin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>100U/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>100&#181;g/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>Invitrogen</td>
			<td>15160054</td>
			<td>10mg/100mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Horse serum</td>
			<td>Invitrogen</td>
			<td>16050122</td>
			<td>5%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SOS Standard Oocyte Solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>746398</td>
			<td>102 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Sigma-Aldrich</td>
			<td>746436</td>
			<td>3 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td>1 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>5 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">External recording solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-methyl-D-glucamine (NMDG)</td>
			<td>Alfa Aesar</td>
			<td>L14282</td>
			<td>115mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>10mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>239232</td>
			<td>2mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>258105</td>
			<td>pH 7.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Internal recording solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-methyl-D-glucamine (NMDG)</td>
			<td>Alfa Aesar</td>
			<td>L14282</td>
			<td>115mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>10mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediamine Tetraacetic Acid (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>E478-500</td>
			<td>2mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>258105</td>
			<td>pH 7.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Labeling solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KOH</td>
			<td>Fisher Scientific</td>
			<td>P250-1</td>
			<td>115mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>10mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>239232</td>
			<td>2mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>258105</td>
			<td>pH 7.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TMR stock solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetramethylrhodamine-5-maleimide (TMR)</td>
			<td>Molcular Probes by Life Technologies</td>
			<td>T6027</td>
			<td>5mM in DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anap stock solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">Anap</td>
			<td>ABZENA (TCRS)</td>
			<td>Custom synthesis TCRS-170</td>
			<td style="width:96px;">1mM in nuclease-free water and 1% NaOH 1N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Material/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pAnap</td>
			<td>Addgene</td>
			<td>48696</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Performance Oocyte Clamp</td>
			<td>Dagan Corporation</td>
			<td>CA-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gpatch Acquisition software</td>
			<td colspan="3">Department of Anesthesiology, University of California, Los Angeles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Analysis software</td>
			<td colspan="3">Department of Anesthesiology, University of California, Los Angeles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recording Chamber</td>
			<td colspan="2">Custom machined</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photo diode detection system</td>
			<td>Dagan Corporation</td>
			<td>PhotoMax-200/PIN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrical shutter driver</td>
			<td>UNIBLITZ</td>
			<td>VCM-D1</td>
			<td></td>
		</tr>
	</tbody>
</table>

voltage-clamp fluorometry in xenopus oocytes using fluorescent unnatural amino acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Figure 4 shows an example of VCF recordings obtained from an oocyte expressing Shaker channels with fast inactivation removed (IR), L382stop-W434F in presence of pAnap and Anap. The W434F mutation blocks the ionic potassium currents, which makes it possible to measure the transient gating charge displacements (gating currents). The simultaneous recordings of gating currents (upper trace) and Anap fluorescence intensity changes (lower trace) upon depolarization demonstrate...

Watch this Scientific Journal Video about Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids at JoVE.com

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

The overall goal of this procedure is to introduce a fluorescent unnatural amino acid into a membrane protein expressed in Xenopus oocytes and to determine the protein's function and structural rearrangement simultaneously, using voltage-clamp fluorometry. This technique will help answer key questions in the field of structure function relations of membrane proteins. Using voltage-clamp fluorometry, we can directly correlate structural rearrangements with protein function.Adding fluorescent unnatural amino acid labeling to it help to overcome previous limitations on the labeling. This was once the accessibility of the labeling sites as well as background labeling due to endogenous binding sites. The implication of this technique extends towards a better biophysical understanding of membrane proteins because you can now probe domains, which previously were inaccessible with traditional voltage-clamp fluorometry.After surgical removal of the Xenopus oocytes, wash the oocytes three times with SOS solution. Select large and healthy oocytes individually, and incubate them at 18 degrees Celsius for at least four hours in Barth's solution supplemented with antibiotics and 5%horse serum before injection. For nuclear injection of DNA, prepare a long and thin injection tip to reach the nucleus without damaging the oocytes.Then, fill the injection tip with oil, and mount the injection tip on the nanoinjector device. Next, install the nanoinjector under a stereo microscope and break the end of the tip with forceps. After that, eject the oil until there is no air bubble trapped inside the end of the tip.Following that, place one microliter of 0.1 microgram per microliter pAnap in nuclease-free water on a piece of Parafilm under the stereoscope, and fill the injection tip with DNA. Then, transfer the oocytes to a mesh-containing injection dish containing Barth's solution supplemented with antibiotics. As the oocyte nucleus is located in the animal pole, aim the injection tip at the center of the animal pole and impale such that the tip reaches near the center of the animal hemisphere.Next, injection 9.2 nanoliters of the pAnap solution into the nucleus of each oocyte. Subsequently, incubate the oocytes in two milliliters of Barth's solution supplemented with antibiotics and 5%horse serum at 18 degrees Celsius for six to 24 hours to allow robust expression of Anap-specific tRNAs and tRNA synthetases. Now, prepare the nanoinjector again, but this time, the injection tip does not need to be as thin as the one for DNA injection.Work in only red light from this point to prevent photobleaching of the Anap. Mix one microliter of one-millimolar Anap with one microliter of one to two micrograms per microliter mRNA directly on a piece of Parafilm, and fill the injection tip with the mix solution. Impale the tip in the vegetal pole just below the membrane, and inject 46 nanoliters of the mix solution in each pAnap-injected oocyte.Then, protect the oocytes from light by placing them in a light-tight box or by wrapping the flask in aluminum foil. Incubate them in Barth's solution supplemented with antibiotics and 5%horse serum at 18 degrees Celsius for two to three days. Replace it with fresh Barth's solution every day, and remove the dead oocytes to avoid contamination.In this setup, the cut-open voltage-clamp equipment is integrated into a fluorescence microscopy setup. The recording chamber is installed on a slider that allows it to move between the standard stereoscope for placing the oocyte and the microscope to perform fluorescence measurements. A photodiode detection system is connected to the C-mount exit port of the fluorescence microscope, and the photocurrent readout is connected to a second input channel in the digital signal processor.A 100-watt, 12-volt halogen lamp is used as a light source for the fluorescence excitation. The excitation is controlled by a mechanical shutter between the excitation light source and the microscope. The activation is triggered via a TTL pulse coming from the digital signal processor that is timed in the recording software such that the shutter opens about 100 milliseconds before the beginning of the recording, and an appropriate filter cube is required in the filter cube turret.For two-color VCF, incubate the prepared oocytes in five-micromolar TMR maleimide in labeling solution for 15 minutes immediately before recording. Then, wash the oocytes with the labeling solution three times to remove excess dye before recording. At this point, the protein is specifically labeled with two different fluorophores.After oocyte mounting, with the animal pole facing upwards and permeabilized, slide the chamber to the microscope and focus on the oocyte using a 4X objective. Next, impale the oocyte with a voltage-sensing V1 electrode. Then, switch to the 40X water immersion objective.Focus on the animal pole, which is facing upwards. After that, turn off the red light. Select the Anap filter cube by turning the filter cube turret and the optical exit port connected to the photodiode.Following that, turn on the halogen lamp at the highest intensity, and open the shutter for two to five seconds to read the background fluorescence intensity originating from the oocyte. Afterward, turn on the clamp, flip the bath/guard switch to active, and adjust the membrane potential to the command potential by turning the knob on the headstage. Subsequently, select the holding potential, step protocol, number, and length of pulses in the recording software.Then, record the voltage-dependent currents and Anap fluorescence intensities. For two-color VCF, select the TMR filter cube by turning the cube turret. Read the background fluorescence for TMR, as described for Anap, and record voltage-dependent currents and TMR fluorescence intensity simultaneously.This figure displays Anap and TMR fluorescence signals using step protocols obtained from the same oocyte expressing a Shaker mutant. By adding a cysteine, accessible from the external solution, into the L382 stop W434F background and labeling it with TMR maleimide, it is possible to probe real-time movements in different regions of the same protein simultaneously. Fluorescence voltage relationship is obtained by plotting the steady-state fluorescence intensity against voltage.After watching this video, you should have a good understanding of how to express fluorescent unnatural amino acids into Xenopus oocytes and how to perform one-color or two-color voltage-clamp fluorometry. Once you master the technique, it should roughly take about the same amount of time as traditional electrophysiology experiments, you just have the extra step of injecting DNA into the nucleus of the oocyte. When attempting this procedure, it's important to remember to check for leak expression in the absence of the unnatural amino acid.This has to be done for every mutation, as the amount of leak expression depends on the insertion site of the stop codon within the protein. This technique now enables researchers to study conformational changes on the cytosolic site of the protein, where many of the regulatory mechanisms occur.

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Research

JoVE Journal

Biochemistry

10.4K visualizações.  University of Montréal. The overall goal of this procedure is to introduce a fluorescent unnatural amino acid into a membrane protein expressed in Xenopus oocytes and to determine the protein's function and structural rearrangement simultaneously, using voltage-clamp fluorometry. This technique will help answer key questions in the field of structure function relations of membrane proteins. Using voltage-clamp fluorometry, we can directly correlate structural rearrangements with protein function.Adding fluore...