Name: how to quantify the fraction of photoactivated fluorescent proteins in bulk and in live cells
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells at JoVE.com

We would like to thank the Dorigo laboratory and the Neuroscience Imaging Service at Stanford University School of Medicine for providing equipment and space for this project.

1. Plasmid Construction
<ol>
	<li>Generate two-color fusion probes. Use a mammalian cell expression vector (see Table of Materials) in which mCherry112 and PA-mCherry113 have been inserted with the restriction sites AgeI and BsrGI.</li>
	<li>Order custom oligo-nucleotides to amplify the monomeric variants of eGFP and PA-eGFP containing the A206K mutation, i.e., mEGFP and PA-mEGFP14 without a stop codon as...

<ol>
	<li>Patterson, G. H., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photoactivatable+GFP+for+selective+photolabeling+of+proteins+and+cells.">A photoactivatable GFP for selective photolabeling of proteins and cells.</a> Science. 297 (5588), 1873-1877 (2002).</li><li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (20), 12651-12656 (2002).</li><li>Renz, M., Lippincott-Schwartz, J., Day, R. N., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+9.">Ch. 9.</a> The Fluorescent Protein Revolution Series in Cellular and Clinical Imaging. , 201-228 (2014).</li><li>Shcherbakova, D. M., Sengupta, P., Lippincott-Schwartz, J., Verkhusha, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocontrollable+fluorescent+proteins+for+superresolution+imaging.">Photocontrollable fluorescent proteins for superresolution imaging.</a> Annual Review of Biophysics. , 303-329 (2014).</li><li>Betzig, E., 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=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313 (5793), 1642-1645 (2006).</li><li>Hess, S. T., Girirajan, T. P., Mason, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high+resolution+imaging+by+fluorescence+photoactivation+localization+microscopy.">Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.</a> Biophysical Journal. 91 (11), 4258-4272 (2006).</li><li>Henderson, J. N., 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=Structure+and+mechanism+of+the+photoactivatable+green+fluorescent+protein.">Structure and mechanism of the photoactivatable green fluorescent protein.</a> Journal of the American Chemical Society. 131 (12), 4176-4177 (2009).</li><li>Subach, F. V., 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=Photoactivation+mechanism+of+PAmCherry+based+on+crystal+structures+of+the+protein+in+the+dark+and+fluorescent+states.">Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (50), 21097-21102 (2009).</li><li>Wiedenmann, 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=EosFP,+a+fluorescent+marker+protein+with+UV-inducible+green-to-red+fluorescence+conversion.">EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (45), 15905-15910 (2004).</li><li>Habuchi, S., Tsutsui, H., Kochaniak, A. B., Miyawaki, A., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mKikGR,+a+monomeric+photoswitchable+fluorescent+protein.">mKikGR, a monomeric photoswitchable fluorescent protein.</a> PLoS One. 3 (12), e3944 (2008).</li><li>McKinney, S. A., Murphy, C. S., Hazelwood, K. L., Davidson, M. W., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bright+and+photostable+photoconvertible+fluorescent+protein.">A bright and photostable photoconvertible fluorescent protein.</a> Nature Methods. 6 (2), 131-133 (2009).</li><li>Shaner, N. 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=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nature Biotechnology. 22 (12), 1567-1572 (2004).</li><li>Subach, F. V., 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=Photoactivatable+mCherry+for+high-resolution+two-color+fluorescence+microscopy.">Photoactivatable mCherry for high-resolution two-color fluorescence microscopy.</a> Nature Methods. 6 (2), 153-159 (2009).</li><li>Zacharias, D. A., Violin, J. D., Newton, A. C., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+lipid-modified+monomeric+GFPs+into+membrane+microdomains+of+live+cells.">Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.</a> Science. 296 (5569), 913-916 (2002).</li><li>Slocum, J. D., Webb, 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=A+Double+Decarboxylation+in+Superfolder+Green+Fluorescent+Protein+Leads+to+High+Contrast+Photoactivation.">A Double Decarboxylation in Superfolder Green Fluorescent Protein Leads to High Contrast Photoactivation.</a> Journal of Physical Chemistry Letters. 8 (13), 2862-2868 (2017).</li><li>Subach, F. V., Patterson, G. H., Renz, M., Lippincott-Schwartz, J., Verkhusha, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+monomeric+photoactivatable+red+fluorescent+protein+for+two-color+super-resolution+sptPALM+of+live+cells.">Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.</a> Journal of the American Chemical Society. 132 (18), 6481-6491 (2010).</li><li>Renz, M., Wunder, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+rulers+to+assess+fluorescent+protein+photoactivation+efficiency.">Internal rulers to assess fluorescent protein photoactivation efficiency.</a> Cytometry A. , (2017).</li><li>Renz, M., Daniels, B. R., Vamosi, G., Arias, I. M., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+the+asialoglycoprotein+receptor+deciphered+by+ensemble+FRET+imaging+and+single-molecule+counting+PALM+imaging.">Plasticity of the asialoglycoprotein receptor deciphered by ensemble FRET imaging and single-molecule counting PALM imaging.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (44), E2989-E2997 (2012).</li></ol>

So far, no method existed to determine in bulk the fraction of PA-FPs expressed in live cells that is photoactivated to be fluorescent. The presented protocol can be used for any spectrally distinct fluorescent protein pair. While exemplified here for the irreversible PA-FPs PA-GFP and PA-Cherry, this approach is in principle applicable to photoconvertible proteins as well. The spectrally distinct fluorescent protein, however, must be selected carefully to minimize spectral overlap given that photoconvertible fluorescent...

In 2002, the first broadly applicable photoactivatable (PA-GFP1) and photoconvertible (Kaede2) fluorescent proteins were described. These optical highlighter fluorescent proteins change their spectral properties upon irradiation with UV-light, i.e., they become bright (photoactivatable fluorescent proteins, i.e., PA-FPs), or change their color (photoconvertible FPs). To date, several reversible and irreversible photoactivatable and photoconvertible fluorescent proteins have been developed3,4. In ensemble or bulk studies, optical highlighters have been used to study the dynamics of entire cells or proteins, and the connectivity of subcellular compartments. Furthermore, optical highlighters enabled single-molecule based superresolution imaging techniques such as PALM5 and FPALM6.
Although the photochemical processes during photoactivation or -conversion have been described for many optical highlighters and even crystallographic structures before and after photoactivation/ -conversion have been made available7,8, the underlying photophysical mechanism of photoactivation and -conversion is not completely understood. Furthermore, thus far only crude estimates exist of the efficiency of photoactivation and -conversion, that is, the fraction of fluorescent proteins expressed that is actually photoconverted or photoactivated to be fluorescent. In vitro ensemble studies have been reported quantifying the shift in absorption spectra and the amount of native and activated protein in a gel9,10,11.
Here, we present a protocol involving fluorescent protein chimeras to assess the fraction of photoactivated fluorescent proteins in bulk and in live cells. Whenever working with genetically encoded fluorescent proteins, the absolute amount of protein expressed varies from cell to cell and is unknown. If one cell expressing a PA-FP shows a brighter signal after photoactivation than another cell, it cannot be differentiated if this brighter signal is due to higher expression of the PA-FP or a more efficient photoactivation of the PA-FP. To standardize the expression level in cells, we introduce internal rulers of genetically coupled spectrally distinct fluorescent proteins. By coupling the genetic information of a photoactivatable fluorescent protein to a spectrally distinct always-on fluorescent proteins, internal rulers are created that will still be expressed to an unknown total amount but in a fixed and known relative amount of 1:1. This strategy allows the quantitative characterization of different UV-light photoactivation schemes, i.e., the assessment of the relative amount of PA-FPs that can be photoactivated with different modes of photoactivation, and thereby permits to define photoactivation schemes that are more effective than others. Furthermore, this strategy allows in principle the assessment of the absolute quantification of the photoactivated PA-FP fraction. To this end, it is important to realize that the presented ensemble studies are intensity-based which makes the analysis more complex as laid out in this protocol. Parameters determining the measured fluorescent intensity, i.e., different molecular brightness, absorbance and emission spectra and FRET effects, need to be considered when comparing fluorescence intensities of different fluorescent proteins.
The presented ratiometric intensity-based quantification of photoactivation efficiency is exemplified for PA-GFP and PA-Cherry in live cells, but is in principle broadly applicable and can be used for any photoactivatable fluorescent protein under any experimental condition.

<table>
	<tbody>
		<tr>
			<td>pEGFP-N1 mammalian cell expression vector</td>
			<td>Clontech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM w/o phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>11054020</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin w/o phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>LabTek 8-well chambers #1.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>12565470</td>
			<td></td>
		</tr>
		<tr>
			<td>Fugene 6</td>
			<td>Promega</td>
			<td>E2691</td>
			<td></td>
		</tr>
	</tbody>
</table>

how to quantify the fraction of photoactivated fluorescent proteins in bulk and in live cells

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells and protein ensembles. Thus far, no method has been available to quantify in bulk and in live cells how many of the PA-FPs expressed are photoactivated to fluoresce.
Here, we present a protocol involving internal rulers, i.e., genetically coupled spectrally distinct (photoactivatable) fluorescent proteins, to ratiometrically quantify the fraction of all PA-FPs expressed in a cell that are switched on to be fluorescent. Using this protocol, we show that different modes of photoactivation yielded different photoactivation efficiencies. Short high-power photoactivation with a confocal laser scanning microscope (CLSM) resulted in up to four times lower photoactivation efficiency than hundreds of low-level exposures applied by CLSM or a short pulse applied by widefield illumination. While the protocol has been exemplified here for (PA-)GFP and (PA-)Cherry, it can in principle be applied to any spectrally distinct photoactivatable or photoconvertible fluorescent protein pair and any experimental set-up.

The protocol presented here shows the ratiometric quantification of the fraction of fluorescent proteins that are photoactivated to be fluorescent (Figure 1). This fraction differs depending upon the mode of photoactivation.
A typical result using short time high-power photoactivation with a confocal laser scanning microscope (CLSM) is shown in Figure 2c. After titratin...

Watch this Scientific Journal Video about How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells at JoVE.com

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

Here, we present a protocol that involves genetically coupled spectrally distinct photoactivatable and fluorescent proteins. These fluorescent protein chimeras permit quantification of the PA-FP fraction that is photoactivated to be fluorescent, i.e., the photoactivation efficiency. The protocol reveals that different modes of photoactivation yield different photoactivation efficiencies.

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells ...

We present here a protocol that involves genetically coupled spectrally distinct photo-activatable and fluorescent proteins.These internal rulers permit quantification of the PAFP fraction that is photo activated to be fluorescent.Thus far, no method has been available to quantify in bulk in life cells how many of the PAFP expressed are photo activated to fluoresce.The presented quantification of photo-activation efficiency is exemplified for PA-GFP and PA Cherry in live cells but it is in principle broadly applicable and can be used for any photo-activatable fluorescent protein under any experimental condition.Whenever working with genetically encoded fluorescent proteins, the absolute amount of proteins expressed varies from cell to cell and it is unknown.To standardize the expression among cells, we introduce internal rulers of genetically coupled spectrally distinct fluorescent proteins.By coupling the genetic information of a photo-activatable florescent protein to a spectrally distinct always on fluorescent protein internal rulers are created that will still be expressed to an unknown total amount but in a fixed known relative amount of one to one.To begin construct the plasmids as described in the accompanying text protocol.Also, culture in the standard cell line in its perspective medium using the phenol red free version.When ready to begin the experiment, harvest the cells using trypsin without phenol red to reduce background florescence.After harvest, fill a 2 ml pipette with the cell-suspension from a confluent cell culture grown in a T 12.5 cell culture flask.Add one drop into each chamber of an eight chamber glass slide.Incubate the slide under standard cell culture conditions for 24 hours.Next transfect the cells using commercial reagents following the distributors protocol.Transfect the cells with the GFP Cherry, PA-GFP Cherry and GFPPA Cherry chimeras.Then return the cells to the incubator to allow for protein expression, folding, and maturation.After a total of 20 hours post transfection, transfer the cells to the stage of a confocal florescent microscope equipped with the humidified and heated environmental chamber pre-warmed to 37

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Biochemistry

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An important first step in any membrane-dependent reaction is binding of protein on the phospholipid membrane. An approach to studying protein interaction with lipid membranes has been developed using fluorescently labeled proteins and flow cytometry. This method allows the study of protein-membrane interactions using live cells and natural or artificial phospholipid vesicles. The advantage of this method is the simplicity and availability of reagents and equipment. In this method, proteins are labeled using fluorescent dyes. However, both self-made and commercially available, fluorescently labeled proteins can be used. After conjugation with a fluorescent dye, the proteins are incubated with a source of the phospholipid membrane (microvesicles or cells), and the samples are analyzed by flow cytometry. The obtained data can be used to calculate the kinetic constants and equilibrium Kd. In addition, it is possible to estimate the approximate number of protein binding sites on the phospholipid membrane using special calibration beads.

Here, we describe a set of methods for characterizing the interaction of proteins with membranes of cells or microvesicles.

In the human body, most of the major physiologic reactions involved in the immune response and blood coagulation proceed on the membranes of cells. An ...

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Author Spotlight: High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...